Investigations Into the Transcriptome of the Toxigenic Marine Dinoflagellate Alexandrium Minutum
Investigations into the transcriptome of the toxigenic marine dinoflagellate Alexandrium minutum
Dissertation
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
-Dr. rer. nat.-
Fachbereich 2 (Biologie/Chemie)
Universität Bremen
vorgelegt von
Ines Yang
September 2009 1. Gutachter: Prof. Dr. Ulrich Bathmann
Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven u.
Universität Bremen
2. Gutachter: Prof. Dr. Dieter Wolf-Gladrow
Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven u.
Universität Bremen Überarbeitete und aktualisierte Version, vorgelegt im Oktober 2010 Contents
1 Summary ...... 1
2 Zusammenfassung ...... 3
3 Introduction ...... 5 3.1 Dinoflagellates – an unusual group of eukaryotes ...... 5 3.2 Phytoplankton Blooms...... 12 3.3 The genus Alexandrium ...... 13 3.4 Paralytic Shellfish Poisoning (PSP) toxins ...... 15 3.5 Allelochemical properties of Alexandrium ...... 20 3.6 Outline of the thesis...... 22
4 Manuscripts ...... 28 4.1 List of Manuscripts ...... 28 4.2 Declaration of contributions ...... 29
5 Manuscript 1...... 30
6 Manuscript 2...... 79
7 Manuscript 3...... 102
8 Manuscript 4...... 116
9 Synthesis...... 175 9.1 Characterisation of the A. minutum transcriptome ...... 175 9.2 Search for sequences associated with PSP toxicity ...... 178 9.3 Transcriptome-level processes associated with growth, nutrient status and stress response...180 9.4 Conclusion...... 182
10 Future Perspectives ...... 185
Appendix ...... 200 I. Manuscript 1, additional file 1: Primer tables and respective PCR conditions ...... 201 II. Manuscript 1, additional file 3: Phyml-based likelihood trees with bootstrap support values205 III. Manuscript 1, additional file 4: Rarefaction curve ...... 222 IV. Manuscript 2, Supplementary table: information on the genes up-regulated at Day 1 or Day 3 of copepod-induced PSP toxin production ...... 224
Danksagung...... 227 Summary 1
1 Summary
Dinoflagellates of the A. minutum species complex are widely distributed bloom- forming, photosynthetic protists typically producing potent neurotoxins which cause paralytic shellfish poisoning (PSP). Human intoxications usually occur via the ingestion of filter-feeding bivalves, but intoxications after consumption of predatory crabs have also been reported. The same group of toxins is produced by certain freshwater cyanobacteria, in which the gene cluster coding for the PSP toxin-producing enzymes has recently been identified. However, the identification of corresponding genes in dinoflagellates is pending, which might be due to their unusual and little understood genomes.
The aim of this thesis is to contribute to understanding gene expression and regulation in dinoflagellates by investigating the transcriptome-level gene expression in A. minutum. The gene expression experiments were based on an expressed sequence tag
(EST) library, which was generated from pooled samples originating from different treatments to include the highest possible diversity of cDNA sequences. A set of microarray probes based on this library was used for different gene expression experiments, such as the comparison of toxic and non-toxic strains. Three stains were compared at two different times of the light phase, yielding a group of 192 genes differentially expressed between toxic and non-toxic strains at both timepoints.
Another experiment was based on the induction of a fivefold increase in toxin levels by the presence of a copepod grazer. In combination with the data obtained from the strain comparison experiment, this resulted in the identification of two sequences potentially correlated to the ability to produce the toxins. Summary 2
Other treatments were designed to represent extreme values of ecologically significant parameters, such as different salinities or nutrient limitation. While the transcriptomic differences between cultures acclimatised to different salinities remained moderate and indicated a high prevalence of differences in post-transcriptional processes, the differences among growth phases and between nutrient-replete and nutrient-limited batch cultures were considerable. By using combinations of comparisons, several genes consistently up-regulated at the transition between exponential growth and stationary phase were identified, as well as 87 genes consistently associated with N- or P- limitation.
In the course of this thesis, I identified several A. minutum genes apparently associated with intracellular toxins, as well as patterns of gene expression indicative of culture growth status and of nutrient limitation. These data substantially add to the emerging field of dinoflagellate transcriptomics. Additionally, they provide a starting point for the potential development of new monitoring tools testing the physiological status or toxin- producing potential of A. minutum populations. Zusammenfassung 3
2 Zusammenfassung
Dinoflagellaten aus dem Artkomplex A. minutum sind weit verbreitete einzellige
Phytoplanktonorgansimen, die häufig Massenvorkommen, sogenannte „Algenblüten“, bilden. Die meisten Stämme und Populationen produzieren starke Nervengifte, die paralytische Muschelvergiftungen (paralytic shellfish poisoning, PSP) verursachen können. Meistens werden diese Vergiftungen durch den Verzehr von Muscheln hervorgerufen, die sich zum Teil von giftbildenden Dinoflagellaten ernährt haben. Auch
Vergiftungen nach dem Verzehr von räuberischen Strandkrabben sind bekannt. Die gleiche Toxingruppe wird auch von im Süßwasser lebenden Cyanobakterien gebildet, in denen vor Kurzem das Gencluster mit den Enzymen des PSP-Toxin-Biosynthesewegs ermittelt wurde. Die Identifizierung der entsprechenden Gene in Dinoflagellaten steht allerdings noch aus, was wahrscheinlich mit deren ungewöhnlichen Genomen zusammenhängt.
Ziel dieser Arbeit ist es, durch Untersuchung der Genexpression von A. minutum auf
Transkriptomebene zum Verständnis der Genexpression und ihrer Regulation in
Dinoflagellaten beizutragen. Grundlage der Genexpressions-Experimente war eine
EST(expressed sequence tag)-Bank, zu deren Herstellung unter verschiedenen
Bedingungen gewachsene Proben kombiniert wurden, um eine möglichst diverse
Population von cDNA- Sequenzen zu erhalten. Anhand dieser Bank wurde ein Katalog von Microarray-Sonden erstellt, der dann für verschiedene Genexpressionsexperimente verwendet wurde. Eins dieser Experimente war ein Vergleich zwischen zwei toxischen und einem nicht toxischen Stamm zu zwei unterschiedlichen Zeitpunkten innerhalb der
Lichtphase. Anhand dieses Versuchs wurde eine Gruppe von 192 Genen identifiziert, deren Expression zu beiden Zeitpunkten signifikant verschieden zwischen den zwei toxischen und dem nicht-toxischen Stamm war. Zusammenfassung 4
Bei einem anderen Experiment wurde die durch die Anwesenheit eines Copepoden hervorgerufene Induktion von fünffach erhöhten Toxinwerten innerhalb der
Alexandrium-Zellen verwendet. In Verbindung mit den Daten aus dem Vergleich zwischen den verschiedenen Stämmen wurden dabei zwei Sequenzen identifiziert, die mit der Toxinproduktion in Verbindung zu stehen scheinen.
In weiteren Versuchen wurden extreme Werte ökologisch relevanter Parameter wie
Salinität oder Nährstoffmangel verwendet. Während die Unterschiede im Transkriptom von an unterschiedliche Salinitäten akklimatisierten Kulturen nicht sehr ausgeprägt waren, aber auf Unterschiede in posttranskriptionellen Prozessen schließen ließen, wiesen Kulturen in unterschiedlichen Wachstumsphasen oder mit unterschiedlicher
Nährstoffversorgung beachtliche Unterschiede auf. Durch Kombination paarweiser
Vergleiche zwischen verschiedenen Wachstumsbedingungen konnten einige Gene identifiziert werden, deren erhöhte Expression mit dem Übergang von exponentieller zu stationärer Wachstumsphase assoziiert waren. Außerdem wurden 87 Gene identifiziert, deren Expression anscheinend spezifisch auf N- oder P-Mangel reagierte.
In dieser Arbeit identifiziere ich sowohl mehrere A. minutum-Gene, die mit dem intrazellulären Toxingehalt in Zusammenhang zu stehen scheinen, als auch charakteristische Genexpressionsmuster für bestimmte Phasen im Kulturzyklus und für
Nährstofflimitation. Diese Informationen sind ein wesentlicher Beitrag zu dem aufkommenden Forschungsfeld der Transkriptomforschung an Dinoflagellaten.
Außerdem sind sie ein Ansatzpunkt für die Entwicklung neuer Systeme zur
Überwachung von Planktonpopulationen, die den physiologischen Zustand oder die
Fähigkeit zur Toxinproduktion in A. minutum-Vorkommen testen könnten. Introduction 5
3 Introduction
3.1 Dinoflagellates – an unusual group of eukaryotes
Dinoflagellates are a highly diverse group of protists that can be found in most aquatic habitats worldwide. Exhibiting a wide diversity of lifestyles, they are often major components of both phytoplankton and micrograzer communities. Other species are benthic photo-, mixo- or heterotrophs, and some are symbionts or parasites of different uni- or multicellular organisms.
Photosynthetic dinoflagellates are among the most important phytoplankton groups in marine ecosystems. While exact data on their global biomass production are not available, they occur from the Arctic and Antarctic marine ice brine channels
(Montresor et al., 2003; Thomson et al., 2006) to the tropics (Parab et al., 2006). In some instances, dinoflagellates can regularly (Reid et al., 1990) or even permanently
(Landry & Kirchman, 2002) dominate the eukaryotic phytoplankton in the marine environment. Although less dominant in freshwater systems, they often contribute significantly to lake phytoplankton Many species are not purely autotrophic but will act as facultative mixotrophs when suitable prey organisms are available (Jeong et al.,
2005b). The “zooxanthellae” of various organisms, such as corals, marine flatworms
(Lopes & Silveira, 1994), certain foraminifera (Lombard et al., 2009) or radiolarians
(Gast & Caron, 1996) are photosynthetic dinoflagellate symbionts.
Similar to the photosynthetic species, heterotrophic dinoflagellates contribute significantly to the heterotrophic plankton worldwide, and can even dominate marine micrograzer communities (Tillmann & Hesse, 1998; Levinsen & Nielsen, 2002; Yang et al., 2004; Vargas & Martínez, 2009). Following an estimate by Dodge (1983), the proportion of heterotrophic dinoflagellates is usually reported to be about 50%. Their Introduction 6 feeding modes can vary considerably (reviewed in Tillmann, 2004). The prey items of the various groups range from bacteria over diatoms and phototrophic or other heterotrophic dinoflagellate species to small metazoa, such as copepod eggs or nauplii.
Some other heterotrophic species are parasites of copepods (Elbrächter, 1988), fish
(Landsberg et al., 1994) or other organisms.
3.1.1 The dinoflagellate genome
The most unusual feature of dinoflagellates is the peculiar organisation of their genome, which is arguably one of the most bizarre eukaryotic machineries known. Both chromosome numbers and DNA content of core dinoflagellate (“dinokaryote”) nuclei are unusually high; depending on the species, haploid dinoflagellate cells can contain
200 or more chromosomes (Shyam & Sarma, 1978). A survey of genome sizes of free- living species revealed DNA contents between 3.6 and 225 pg DNA per cell, which corresponds to roughly 1 to 75 times the haploid human value (LaJeunesse et al., 2005).
Most of this DNA is permanently condensed into a liquid crystalline state (Livolant &
Bouligand, 1978), which is stabilised by high concentrations of bivalent cations (Levi-
Setti et al., 2008). This arrangement lacks histones and consequently nucleosomes, but the nuclei contain low abundances of basic histone-like proteins that were acquired by horizontal gene transfer from proteobacteria (Moreno Díaz de la Espina et al., 2005;
Chan et al., 2006). The DNA itself contains high amounts of modified bases. In addition to the methylation of cytosine, which is common in other eukaryotes as well, between
12 and 68% of the dinoflagellate thymine is replaced by the unusual base hydroxymethyluracil.
While dinoflagellates contain the largest known protist transcriptomes - A. tamarense was estimated to have about 40,000 transcribed genes, and evidence for extensive genome duplication coupled with chromosome reorganisation has been reported (Zhang Introduction 7 et al., 2009; Moustafa et al., 2010) - their gene content is considerably lower than would be expected based on genome scale. Their genome sizes appear uncoupled from gene numbers (Moustafa et al., 2010).
The combination of these features has been interpreted as indicative of high amounts of non-coding structural DNA. This is supported by the restriction of transcriptional activity to DNA filaments extending from the main body of the chromosome into the nucleoplasm (Sigge, 1984), as well as by the results of the first available whole genome sequence survey of a dinoflagellate (McEwan et al., 2008). About 90% of the examined
Heterocapsa triquetra sequences were found to be apparently random, non-repetitive
DNA (McEwan et al., 2008).
Many dinokaryote genes are organised into large gene families with evidence for expression correlation among family members (Moustafa et al., 2010), and highly expressed genes tend to be organised in tandem repeats (Bachvaroff & Place, 2008).
Uniquely, many genes show signs of being descended from processed mRNAs re- integrated into the genome, apparently having been cycled through an mRNA intermediate once or several times during recent evolutionary history (Slamovits &
Keeling, 2008).
For most or even all transcribed genes, mRNA maturation involves a trans-splicing process in which a common spliced leader (SL) sequence is added to the 5’-end of mature mRNAs (Lidie & Van Dolah, 2007; Zhang & Lin, 2009). Unlike most other core dinoflagellate peculiarities, SL trans-splicing has also been shown in earlier-diverging lineages such as the syndineans (Bachvaroff et al., 2009) and perkinsids (Zhang et al.,
2007; Joseph et al., 2010) (see below). Introduction 8
The unusual genomic setups, as well as some open questions regarding gene family structure and the regulation of gene expression, complicate the interpretation of genomic studies on dinoflagellates, but also reinforce the interest in such projects.
3.1.2 Evolutionary origin
Phylogenetically and systematically, dinoflagellates are one of the three main groups comprising the alveolates, the other two being the predominantly parasitic apicomplexa and the usually heterotrophic ciliates.
The earliest diverging group of the dinoflagellate lineage are the perkinsids (Gómez et al., 2009; Hoppenrath & Leander, 2009), intracellular parasites of various marine
(Moreira & López-García, 2003; Stelter et al., 2007) and freshwater organisms (Lepère et al., 2008). The perkinsids are sister to a group of three crown clades of unresolved branching order: the “Marine Alveolate Group I”, the “Marine Alveolate Group II” also known as syndinean dinoflagellates and the core dinoflagellates which in this context are called “dinokaryotes” (Guillou et al., 2008; Hoppenrath & Leander, 2009;
Skovgaard et al., 2009) (Fig.1). Both the syndineans and the Marine Alveolate Group I encompass relatively few known species, all of which appear to be parasites, and a vast diversity of organisms known only from environmental sequence surveys from a wide range of marine environments (Groisillier et al., 2006; Dolven et al., 2007; Guillou et al., 2008; Skovgaard et al., 2009).
While the physiology and indeed anatomy of most Group I and Group II alveolates remains elusive, the intracellular lifestyle of the perkinsids is reminiscent of that of derived apicomplexan parasites, and like apicomplexans they use an apical complex to access the cytoplasm of the host cell (reviewed in Leander & Keeling, 2003). By contrast the first-diverging apicomplexan clade, the colpodellids, are small free-living predatory flagellates, which use their apical complex in the myzocytosis of prey cells Introduction 9
(Leander et al., 2003) (Fig 1). This feeding mode, which is also known from dinoflagellates (Calado et al., 1998; Vogelbein et al., 2002) and consists of the ingestion of prey cytoplasm through a perforation in the periplast, is thought to be ancestral within the clade containing apicomplexans, perkinsids and dinoflagellates (Leander &
Keeling, 2003).
The available evidence suggests that this ancestor additionally contained a plastid that gave rise to both the dinoflagellate peridinin plastids and its highly reduced apicomplexan counterpart, the apicoplast. While dinoflagellates contain many heterotrophic lineages, both the early-diverging non-photosynthetic dinoflagellate
Crypthecodinium cohnii (Sanchez-Puerta et al., 2007) and the perkinsid Perkinsus marinus (Stelter et al., 2007) were shown to contain plastid-derived genes and probably even cryptic plastids. The recently discovered sister taxon of apicomplexa, the
Chromerida with the only known species Chromera velia, is photosynthetic and contains a plastid which is morphologically similar to those of peridinin-containing dinoflagellates (Moore et al., 2008). Like the plastid of an even earlier branching photosynthetic alveolate which is only known by its strain designation CCMP3155, the
Chromera plastid contains several morphological and genomic features retained in either apicomplexan or dinoflagellate plastids. The plastid genomes of the two basal lineages combine the genes found in the either apicomplexan or dinoflagellate plastid genomes (Janouškovec et al., 2010).
The origin of this joint plastid remains under debate. The chromalveolate hypothesis
(Cavalier-Smith, 2003) states that cryptomonads, haptophytes, stramenopiles and alveolates can be traced back to one chromalveolate ancestor which acquired a rhodopyhte as secondary endosymbiont. All non-photosynthetic members in these groups, such as the ciliates, are postulated to have subsequently lost their plastids. Introduction 10
Fig. 1: Evolutionary history of Apicomplexa. Modified after Oborník et al. (2009).
*Ichthyodinium chabelardi, semi-schematic drawing of motile cell, Skovgaard et al.
(2009). Introduction 11
While some phylogenies provided moderate support for a sister group relationship of the dinoflagellate peridinin plastids with haptophyte chloroplasts, apparently suggesting serial endosymbiosis (Bachvaroff et al. %RG\á et al., 2009), whole-plastid genome phylogenies of Chromera and CCMP3155 supported a close relationship to the plastids of heterokonts. The picture is further complicated by supertree phylogenies indicating that chromalveolates might not be monophyletic, as haptophytes and cryptomonads might be more closely related to plants than to alveolates and heterokonts, while the primarily non-photosynthetic Rhizaria cluster close to stramenopiles (Burki et al., 2008; Lane & Archibald, 2008; Reeb et al., 2009).
From a paleontological point of view, the oldest evidence of chemicals usually regarded as biomarkers for dinoflagellates was found to be associated with early Cambrian acritarchs (~520 million years ago). As acritarchs is a combined group for organic- walled microfossils that cannot be definitely allocated to known organisms, the presence of an early dinoflagellate ancestor in this group seems plausible. However, small amounts of the biomarkers in question have also been found in extant diatoms and prymnesiophytes (Volkman et al., 1990; Volkman et al., 1993), so their occurrence in fossil organisms not related to dinoflagellates cannot be ruled out.
The oldest known cysts that can be unambiguously allocated to dinoflagellates occur in mid-Triassic deposits at about 240 million years before present (Fensome et al., 1996;
John et al., 2003), at the end of a 130 million year “phytoplankton blackout” lasting from the Devonian/Carboniferous boundary until the late Triassic. This gap in the phytoplankton record was probably caused by processes linked to the assembly of all continents into the single land mass Pangaea. The extensive loss of shelf seas, differences in global nutrient flows and other oceanographic and climatic effects are Introduction 12 likely to have caused a substantial reduction in phytoplankton primary production leading to the extinction of most of the older acritarch lineages (Riegel, 2008).
After the break-up of Pangaea and the return of shelf seas, the Mesozoic was characterised by the radiation of dinoflagellates, coccolithophorids and diatoms. The ongoing success of these “red lineage” groups has been linked to the different trace element requirements of red and green plastids. The “red lineage” is supposed to be slightly better adapted to the low availability of Fe and Mn in highly oxidised oceans, which may have helped them to attain dominance during the Mesozoic phytoplankton radiation (Quigg et al., 2003; Falkowski et al., 2004).
Tracing the development of sea levels and shelf sea areas, the diversity of cyst- producing dinoflagellates rapidly increased during the Upper Triassic (Sluijs et al.,
2005; Palliani & Buratti, 2006), reached maxima in the Jurassic (MacRae et al., 1996), the Cretaceous and the early Eocene, after which cyst diversity started a long-term decline to the present values. This might be attributed to the relatively high wind speeds, thermohaline circulation and increased upper- ocean turbulence in the
Quaternary, which lead to the present predominance of diatoms in oceanic systems
(MacRae et al., 1996; Sluijs et al., 2005). However, dinoflagellates still remain a major part of planktonic systems, and regularly outcompete diatoms under low turbulence, stratified water conditions or under silicate limitation.
3.2 Phytoplankton Blooms
Many planktonic organisms can form mass occurrences in the water column. When the cell densities reach values considerably higher than their general background distribution, they are called blooms (see Smayda, 1997). Blooms can be almost monospecific, others are formed by a combination of species (Garcés et al., 2005;
Popovich et al., 2008) . Many prominent blooms can be traced back to high nutrient Introduction 13 loads (Hallegraeff, 1993; Hwang & Lu, 2000; Yentsch et al., 2008), but they can occur whenever a species is able to outgrow its competitors while partially reducing grazer pressure (Irigoien et al., 2005). Some dinoflagellates bloom in unexpected places, such as below the Baltic winter ice cover (Spilling, 2007). Others are more benthic than planktonic, like the palytoxin-producing Ostreopsis ovata (Mangialajo et al., 2008).
As the base of the marine food chains many phytoplankton blooms are beneficial for ocean productivity and hence benefit the fishing and shellfish industries (Hallegraeff,
1993). Other blooms however, most often of dinoflagellates, can significantly disrupt ecosystems (Wear & Gardner, 2001), harm commercially important species (Tang &
Gobler, 2009) or produce substances toxic to humans (Van Dolah, 2000). Coastal eutrophication may in some cases lead to an increase in such harmful bloom events.
Under nitrate or phosphate limited conditions, many diatom species can outcompete dinoflagellates due to generally faster nutrient uptake capabilities and subsequently higher growth rates. When the concentrations of these macronutrients are increased, diatom growth is likely to be limited by silicate, increasing the likelihood of dinoflagellate dominance (Smayda, 1990; Smayda, 1997). While part of the apparent global increase of harmful algal blooms (HABs) might be due to increased scientific awareness and increased commercial activities in coastal areas, eutrophication, climatic effects and the introduction of new harmful species by ship ballast water or import of shellfish stocks for aquaculture are likely to alter the current patterns of bloom occurrence (Hallegraeff, 1993; Scholin et al., 1995; Nagai et al., 2007; Leung &
Dudgeon, 2008).
3.3 The genus Alexandrium
The gonyaulacoid dinoflagellate genus Alexandrium is reported to contain 28 species
(Gómez, 2005), 11 of which are recognised as harmful, according to the IOC Introduction 14
Taxonomic Reference List of Toxic Algae (Moestrup, 2004). However, the determination of biologically meaningful species’ boundaries is still in progress, and some of the listed species are probably conspecific or contain groups of several cryptic species (see below). Alexandrium species are marine, occur predominantly in coastal habitats and contain chloroplasts. They can apparently all survive as purely photosynthetic organisms in culture, but at least some members are known to be facultatively mixotrophic (e.g. Jeong et al., 2005b).
According to molecular clock calculations by John et al. (2003), the genus Alexandrium probably originated around 77 million years ago, in the late Cretaceous, with confidence limits extending up to 119 million years ago (mid-Cretaceous). Each of these dates would coincide with one of the three all-time peaks in dinoflagellate cyst diversity, two of which are reported for mid- and late Cretaceous assemblages (Sluijs et al., 2005).
The A. tamarense species complex, the most well-studied group within the genus, most likely originated during the early Neogene (23 million years ago, with confidence limits until the late Palaeogene, 45 million years ago). John et al. (2003) suggest that its radiation might have be determined by plate movements, oceanographic changes and climatic fluctuations, leading to apparent geographic clades in small- and large-subunit rDNA-based phylogenies (Scholin et al., 1995; John et al., 2003). Group affiliation was highly correlated with toxicity; all tested members of three of the groups were nontoxic, whereas every tested isolate of the remaining two groups was toxic (John et al., 2003;
Lilly et al., 2007).
3.3.1 The Alexandrium minutum species complex
A similar pattern was discovered in A. minutum, the type species of Alexandrium. A review of the morphological and phylogenetic diversity among several closely related species revealed that A. lusitanicum and A. angustitabulatum could not be distinguished Introduction 15 from A. minutum, whereas A. tamutum, A. insuetum and A. andersonii were shown to be morphologically and phylogenetically distinct (Montresor et al., 2004; Lilly et al.,
2005). The A. minutum species complex was shown to consist of two major clades: a
“Pacific clade” containing strains from New Zealand, Taiwan, Malaysia and Vietnam
(Lilly et al., 2005; Lim et al., 2007), and a “Global clade” consisting of strains from
Europe, South Africa and Australia. Within each clade, strains clustered according to origin, although the diversity within the Global clade could only be revealed by microsatellite analysis (McCauley et al., 2009).
The ecological niche of A. minutum was extensively investigated along the Catalan coast (Bravo et al., 2008). In this region, A. minutum cyst beds are present in all sampled harbours and at many beaches. The most important factor in determining the capacity for blooms was the available nitrogen source: A. minutum was dominant at high NO3-levels, whereas additional high NH4-levels favoured the co-occurring “A. catenella” populations.
3.4 Paralytic Shellfish Poisoning (PSP) toxins
Of the 11 toxic Alexandrium species listed by the Moestrup et al. (2004), 8 are reported to be associated with PSP toxin production. These tetrahydropurine neurotoxins form a group of more than 20 known analogues, the most intensively studied of which are probably saxitoxin and the gonyautoxins (GTX) (reviewed in Krock et al., 2007). They act by selectively inhibiting sodium channels, which leads to the potentially severe human intoxication syndrome called PSP (Kao & Walker, 1982). Typical symptoms are paraesthesia, followed by distinct muscular weakness, loss of motor coordination, and paralysis. In severe cases or without adequate treatment, victims may die from respiratory failure (Lehane, 2000; García et al., 2004). Most intoxications result from consumption of filter-feeding bivalves, which tend to accumulate the toxins after Introduction 16 feeding on PSP producers. However, toxins can also be propagated through the food chain, and intoxication through the ingestion of predatory invertebrates such as crabs is possible. Due to widespread resistance of marine invertebrates against PSP toxins, in both cases the resulting concentrations can be extremely high (Llewellyn et al., 2002;
Anderson et al., 2005). Apart from the detrimental effects on humans, PSP-induced deaths of marine animals such as whales or seabirds are also occasionally reported
(Geraci et al., 1989; Trainer & Baden, 1999; Shumway et al., 2003; Doucette et al.,
2006).
Fig. 2: Naturally occurring PSP toxins, including carbamoyl, N-sulfocarbamoyl and decarbamoyl derivatives. Saxitoxin = STX; neosaxitoxin = NEO; gonyautoxins 1,2,3,4
= GTX 1,2,3,4; B1 (=GTX5); B2 (=GTX6); dc = decarbamoyl toxins. Modified after
Krock et al. (2007). Introduction 17
In order to prevent PSP outbreaks, extensive monitoring programmes are indispensable.
While these programmes ensure consumer safety, monitoring alone can do little to mitigate the economic effects of harmful algae occurrences. The necessary closure of shellfish harvesting areas whenever exposure exceeds safe limits, which is often followed by depression of consumer demand in the affected regions as well as with negative impacts on tourism industries, regularly leads to considerable economic losses
(Lehane, 2000). HAB forecasting projects aim to decrease these impacts (e.g. NOAA
Harmful Algal Bloom Forecast System, http://tidesandcurrents.noaa.gov/hab/; CSCOR: http://www.cop.noaa.gov/news/fs/ne_hab_2009.html) by providing seasonal and short- term forecasts based on data from oceanographic measurements, weather forecasts, cyst beds and monitoring results.
3.4.1 Toxin variability in A. minutum
Toxin content in A. minutum can vary substantially, and both toxic and non-toxic strains co-exist in both the global and the Pacific clade (Lilly et al., 2005; McCauley et al.,
2009). Along the Irish coast, toxic and non-toxic A. minutum populations are reported to occur in different regions (Touzet et al., 2007), but other populations contain otherwise indistinguishable toxic and non-toxic strains at the same time (McCauley et al., 2009).
While the toxin profile of most A. minutum strains is dominated by gonyautoxins, even strains with identical LSU rDNA sequences can contain markedly different profiles
(Hansen et al., 2003). Unlike strains from other locations, those of New Zealand isolates were reported to contain substantial amounts of saxitoxin and neosaxitoxin (Chang et al., 1997; MacKenzie & Berkett, 1997). A toxic Taiwanese strain and several other strains from the same region had very typical A. minutum toxin profiles dominated by
GTX4 and GTX1 (Hwang & Lu, 2000; Chou et al., 2004). Introduction 18
3.4.2 Biosynthesis of PSP toxins
Apart from Alexandrium species, PSP toxins are also produced by the dinoflagellates
Gymnodinium catenatum and Pyrodinium bahamense var. compressum, as well as by various freshwater cyanobacteria (Moustafa et al., 2009; Stucken et al., 2010). This unusual distribution lead to the hypothesis that PSP toxin production in dinoflagellates might be due to symbiotic bacteria (e.g. Gallacher et al., 1997; reviewed in Töbe, 2003).
However, several studies demonstrated that removal of bacteria from Alexandrium cultures did not eliminate toxin production (Lu et al., 2000; Hold et al., 2001; Uribe &
Espejo, 2003). The situation is complicated by bacterial production of non-PSP compounds which can mimic PSP toxins in HPLC analysis (Baker et al., 2003; Prol et al., 2009).
PSP toxin profiles are usually strain specific but stable within one strain (Cembella &
John, 2006). Profile inheritance follows a Mendelian segregation pattern (Ishida et al.,
1998) indicative of the involvement of nuclear-coded enzymes. Several such interconverting enzymes have been characterised on the protein level. Based on crude cell extracts, Oshima (1995) characterised an A. tamarense oxidase transforming GTX 3 to GTX 4 and a G. catenatum N-sulfotransferase forming C2 from GTX3. The same sulfotransferase activity was observed in another toxic and a non-toxic G. catenatum isolate. In a later study, a similar enzyme was found in a crude enzyme extract of A. tamarense (Wang et al., 2007). Sako et al. (2001) purified and characterised another
PSP toxin sulfotransferase from G. catenatum. An O-sulfotransferase specifically transferring a sulfonyl group to O-22 of 11-hydroxy-STX to produce GTX 3 was purified and characterized by Yoshida et al. (2002). Based on the very specific activity of this enzyme, which specifically transfers a sulfate residue to a carbamoyl group present only in some saxitoxin analogues, the authors suggested that STX might be the Introduction 19 only toxin directly synthesized. Then all other PSP toxins might be derived by a fixed set of interconversions (Fig. 3).
Fig. 3: Transformation of PSP toxins after Yoshida et al. (2002). Enzymes characterized by Sako et al. (2001) (orange ovals), Yoshida et al. (2002) (blue box) and
(Wang et al., 2007) (green rectangle). Introduction 20
While the data mentioned above combine to a comprehensive hypothesis of the biochemical processes determining the dinoflagellate PSP toxin profiles, the PSP toxin synthesis pathway is largely assumed to be identical to the cyanobacterial one, which is usually explained by horizontal gene transfer (Shimizu, 1996; Kellmann et al., 2008a).
The cyanobacterial pathway and the corresponding gene cluster have recently been identified in Cylindrospermopsis raciborskii T3 (Kellmann & Neilan, 2007; Kellmann et al., 2008b). This cluster contains >35 kb of sequence coding for 26 proteins; PSP toxin synthesis is reported to be initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase. Similar clusters were found in other PSP toxin-producing cyanobacterial species (Mihali et al., 2009), and further studies suggested that at least parts of this cluster have been frequently laterally transferred between different cyanobacterial genera (Kellmann et al., 2008a). Conclusive evidence of the homology of this gene cluster to dinoflagellate sequences is pending.
3.5 Allelochemical properties of Alexandrium
In addition to the rather well characterised toxins, Alexandrium species produce less well understood extracellular compounds which cause negative effects against other protists and which are only now in the process of being chemically characterised (Ma et al., 2009). These allelochemicals apparently act by attacking the outer cell membrane of target species, leading to cell deformation and eventually lysis (Tillmann & John, 2002).
Susceptible organisms include potential grazers, like the predatory ciliate
Rimostrombidium caudatum and various heterotrophic dinoflagellates, as well as autotrophic competitors (see Tillmann et al., 2008). Similar allelochemicals have been found in other dinoflagellate species, such as Peridinium aciculiferum or Fragilidium subglobosum (Rengefors & Legrand, 2007; Tillmann et al., 2008). In the case of the karlotoxin produced by Karlodinium veneficum, the substances apparently targeted at Introduction 21 protistan grazers can also exhibit cyto- and ichthytoxic properties and are thus officially recognised as toxins (Adolf et al., 2007).
Independently of both species-specific and strain-specific PSP toxin content, allelochemical activity in Alexandrium is found in all species tested but can differ substantially between strains even within one A. minutum population. As activity between strains of different species varied widely among donor/target combinations, different Alexandrium species might produce different compositions of allelochemically active compounds (Tillmann & John, 2002; Tillmann et al., 2008; Tillmann et al.,
2009).
While allelochemical interactions are usually discussed in the light of population-scale processes such as competition and bloom formation (reviewed in Legrand et al., 2003;
Granéli & Hansen, 2006), a recent simulation study suggests that their primary and possibly evolutionarily favoured functions might be predator-prey or host-parasite interactions mediated by a “chemical envelope” of allelochemical substances around each individual algal cell (Jonsson et al., 2009). Independent of cell density, these envelopes might act as grazer or parasite deterrent, lyse prey cells or cause leakage of host cells during “casual parasitism”. In this model, any bloom-promoting function of allelopathic interactions would be a side effect which can only set in after bloom formation of the producing species is well under way (Jonsson et al., 2009). This might explain the low incidence of non-lytic clones within an A. tamarense population identified by Tillmann et al. (2009). The same authors also found a high intra-strain variability in lytic potency, indicating a low evolutionary pressure on the quantitative aspect of lytic activity apparently resulting from an intermittantly reduced or inconsistent evolutionary pressure for high allelochemical activity within the “chemical cloud” of local A. tamarense aggregations. Introduction 22
Once cell densities in a developing Alexandrium bloom are high enough to produce a
“chemical cloud”, allelochemical interactions can effectively change the plankton community. When natural plankton samples were exposed to cell-free filtrates of an A. tamarense culture at bloom concentration (corresponding to 34,000 cells l-1), overall growth rate was considerably reduced. Especially ciliates, nanoflagellates and the autotrophic dinoflagellate Scrippsiella trochoidea were inhibited, whereas the cell numbers of small dinoflagellates were positively affected and bacterial numbers increased considerably (Fistarol et al., 2004). This might imply a direct or indirect enhancement of putative prey organisms for the facultative mixotroph Alexandrium
(Jeong et al., 2005b) in addition to the repression of grazers and competitors. Even if these effects in dense Alexandrium populations are unlikely to convert direct evolutionary advantages to specific clones due to the probably rapid dissipation of related cells (Jonsson et al., 2009), they are still a major ecological factor promoting the formation of even denser blooms as well as their maintenance.
3.6 Outline of the thesis
The aim of this thesis was to obtain transcriptome-level information on processes associated with PSP toxin production or with bloom-relevant processes such as growth, allelochemical activity, nutrient limitation, or stress, for the dinoflagellate Alexandrium minutum.
3.6.1 Rationale
A. minutum is a typical toxin-producing harmful algal bloom species (see Chang et al.,
1997; Hwang & Tsai, 1999; Bravo et al., 2008). Although it does not achieve the high toxin cell quotas of the A. tamarense species group, the availability of toxic and non- toxic strains from the same population, which are similar enough to contain identical
LSU and SSU rDNA sequences, recommend this species as a model for the study of Introduction 23
PSP toxin production. Due to its fast growth rate of up to one division per day, and to the availability of numerous published studies on its physiological reaction to a wide range of growth conditions, it also is a convenient model to investigate other bloom- related processes.
While many studies have investigated physiological phenomena associated with PSP toxin content and toxin production in different Alexandrium species (Flynn et al., 1994;
Taroncher-Oldenburg et al., 1999; Hwang & Lu, 2000), only fragmentary data exist on the associated biochemical pathway in dinoflagellates. A hypothetical pathway based on labelled precursor studies carried out mainly in cyanobacteria (Shimizu et al., 1984;
Shimizu, 1996) was shown to be largely accurate for prokaryotic PSP toxin producers
(Kellmann & Neilan, 2007; Kellmann et al., 2008a), but the corresponding genes and enzymes in dinoflagellates remain elusive. Only a pathway for the conversion of saxitoxin into the other PSP toxins has been identified in dinoflagellates to date (Sako et al., 2001; Yoshida et al., 2002; Wang et al., 2007).
Both the elucidation of the dinoflagellate PSP toxin pathway and the development of screening methods to assess the physiological status of natural populations of harmful species are bound to be greatly facilitated by the availability of sizeable molecular datasets for the species in question. The current method of choice for the production of such datasets is large-scale sequencing of the genome or the transcriptome of the study organism, which enables both the detection of conserved genes of known function and the discovery of new genes which can than be characterised based on sequence properties and expression analysis.
3.6.2 Characterisation of the A. minutum transcriptome
In non-model organisms without available genome sequences, projects aimed at the investigating of gene expression are usually based on a transcriptomic databases (e.g. Introduction 24
Okamoto & Hastings, 2003; Lidie et al., 2005; Van Dolah et al., 2007). Genome sequencing in dinoflagellates has so far proven extremely challenging, which is presumably due to their enormous size and unusual structure. While some sequencing initiatives are under way, no sequenced genome is available to date. However, given the large amounts of presumably “gene-empty” structural DNA (McEwan et al., 2008) and a high prevalence of pseudogenes (Bachvaroff & Place, 2008), even a closed genome might be less useful for the investigation of actively transcribed genes than a carefully composed transcriptomic library.
Manuscript 1 describes the construction and characterisation of an A. minutum EST library, the first genomic database for its species. Similar libraries are published for other Alexandrium species (Hackett et al., 2005; Jaeckisch et al., 2008; Uribe et al.,
2008; Toulza et al., 2010), but none of these are based on a comparable number of physiological conditions. As the investigation of gene expression is greatly facilitated by the availability of mRNA sequence data, the highly inclusive EST library introduced in Manuscript 1 is a very important resource for the study of transcriptomic responses of
Alexandrium to different physiological and environmental conditions. These data were used to develop a microarray probe set for further gene expression analyses in A. minutum, the first sets of which are detailed below.
3.6.3 Search for sequences associated with PSP toxicity
The ability to synthesize the tetrahydropurine neurotoxins which cause paralytic shellfish poisoning is not unique to dinoflagellates, but also found in freshwater cyanobacteria (e.g. Stucken et al., 2010). This led to the hypothesis that the dinoflagellate ability to produce these toxins might be a result of inter-kingdom lateral gene transfer (Shimizu, 1996; Kellmann et al., 2008a). Given the high prevalence of mixotrophy in dinoflagellates (Jeong et al., 2005b) in combination with their high Introduction 25 capacity to include new sequences such as organellar genes (Bachvaroff et al., 2004;
Nash et al., 2007) or reverse-transcribed processed mRNAs (Slamovits & Keeling,
2008) into their genomes, the implied independent incorporation events into the genomes of several non-related dinoflagellate clades are not inconceivable. Following the recent elucidation of the PSP toxin gene cluster in the cyanobacterium
Cylindrospermopsis raciborskii (Kellmann et al., 2008b), the identification of dinoflagellate homologues based on sequence similarity should be trivial.
In the second part of Manuscript 1, these sequence similarity-dependent methods are combined with a microarray-based screening approach aimed to identify reproducible gene expression differences between toxin-producing and non-toxin-producing strains.
The latter approach primarily measures differences in gene-specific mRNA abundances between pairs of strains, without discriminating between toxicity-related differences and toxin-unrelated variability. A combination of several such comparisons resulted in a manageable list of candidate genes that can be described as potentially associated with
PSP toxin-related processes.
Several physiological or ecological functions of PSP toxins have been suggested, among others nitrogen storage (reviewed in Cembella, 1998), activity as pheromones
(Wyatt & Jenkinson, 1997), and influences on the dinoflagellate-associated bacteria in the phycosphere (Jasti et al., 2005). The hypothesis that PSP toxicity in dinoflagellates is linked to deterring effects on copepod grazers (Turner & Tester, 1997) was supported by an increase in A. minutum PSP toxin production in response to waterborne signals from certain copepod predators, which was correlated to an increased resistance of A. minutum to grazing by this copepod (Selander et al., 2006). Bergkvist et al. (2008) found this effect to be proportional to the intrinsic toxicity of the different A. minutum Introduction 26 strains. Depending on the strain and the copepod tested, they report responses of up to
20 times higher toxin concentrations.
This induction is a highly convenient model to investigate the A. minutum genes associated with toxicity levels. A comparison of induced versus non-induced cultures will yield relative expression levels correlated with different levels of PSP toxicity within the same strain. The presence of predator cues is likely to elicit less severe physiological responses, and consequently a much more narrowly targeted gene expression signal, than other treatments known to influence cellular toxin content, such as nutrient limitation or suboptimal growth conditions. Manuscript 2 describes such a comparison between induced and non-induced cultures and relates the identified differences in gene expression to the differences between toxic and non-toxic strains found in Manuscript 1. Significantly, two of the sequences identified as higher expressed in toxic than in non-toxic A. minutum in Manuscript 1 were also found to be up-regulated in association with the induction of higher toxin content.
3.6.4 Transcriptome-level processes associated with growth, nutrient status and
stress response
A. minutum is a eurythermal and euryhaline species which can bloom at a wide variety of temperatures and salinities (Vila et al., 2005; Bravo et al., 2008) and can achieve high growth rates at a wide range of light conditions (Hwang & Lu, 2000). The outbreak of A. minutum blooms is reported to be mainly determined by nitrate availability, irrespective of temperature or time of the year. The densest blooms usually occur at a combination of very high nitrate levels and elevated phosphate values (Bravo et al.,
2008), implying that under natural conditions A. minutum growth is mostly nutrient- limited. As nutrient input is often associated with freshwater runoff, elevated nutrient levels tend to coincide with changes in salinity. Optimal bloom salinities at the Introduction 27
Mediterranean coast were reported to be around 34-36 on the practical salinity scale
(Bravo et al., 2008).
Manuscripts 3 and 4 deal with the physiological and transcriptome-level responses of A. minutum to reduced and elevated salinities (Manuscript 3), in exponential or stationary growth phases and under nutrient starvation (Manuscript 4). In both studies physiological processes related to bloom development or maintenance, such as growth, allelochemical activity, PSP toxin contents and intracellular nutrient levels or acclimatisation to new salinity levels are combined with gene expression data comparing the respective treatments with exponentially growing control cultures.
In addition to contributing a new level of data to the understanding of A. minutum physiology, I examine the extent of differential mRNA abundances during these treatments, which adds new data to the ongoing discussion on the distinctive characteristics of dinoflagellate gene expression. Manuscripts 28
4 Manuscripts
4.1 List of Manuscripts
Manuscript 1: Comparative Gene Expression in toxic versus non-toxic Strains of the marine Dinoflagellate Alexandrium minutum. (I. Yang, U. John, S. Beszteri, G.
Glöckner, B. Krock, Alexander Goesmann, A. Cembella). BMC Genomics 11(1): 248
Manuscript 2: Grazer induced toxin formation in dinoflagellates: A transcriptomic model study. (I. Yang, E. Selander, H. Pavia, U. John). European Journal of Phycology, accepted with minor revisions
Manuscript 3: Physiological and Gene Expression Responses to Salinity Stress in
Alexandrium minutum (I. Yang, S. Beszteri, U. Tillmann, A. Cembella, U. John)
Submitted for the Proceedings of the 13th International Conference on Harmful Algae
Manuscript 4: Growth- and limitation-related gene expression in Alexandrium minutum
(I. Yang, S. Beszteri, U. Tillmann, A. Cembella, U. John). To be submitted. Manuscripts 29
4.2 Declaration of contributions
Manuscript 1: The experiments were planned together with U. John, and the treatments included in the EST library were carried out together with S. Bestzeri. The in-house part of the bioinformatics analyses was carried out by the candidate, who also planned and carried out the strain comparison experiment and drafted the manuscript.
Manuscript 2: The experiment was planned together with E. Selander. The candidate carried out the genomic part of the experiment and the corresponding analysis and drafted substantial parts of the manuscript.
Manuscript 3: The experiment was planned together with U. John. The candidate was in charge of the physiological experiments, carried out the genomic part, analysed the data and drafted the manuscript.
Manuscript 4: The experiment was planned together with U. John and U. Tillmann.
The candidate was in charge of the physiological experiments, carried out the genomic part, analysed the data and drafted the manuscript. Manuscript 1 30
5 Manuscript 1 Manuscript 1 31
Comparative Gene Expression in toxic versus non-toxic
Strains of the marine Dinoflagellate Alexandrium minutum
Ines Yang1,2, Uwe John1§, Sára Beszteri1, Gernot Glöckner3,4,5, Bernd Krock1,
Alexander Goesmann6 and Allan Cembella1
1 Alfred-Wegener-Institut für Polar- und Meeresforschung, Am Handelshafen 12, 27570
Bremerhaven, Germany
2 School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK
3 Fritz Lipmann Institute for Age Research, Beutenbergstraße 11, 07745 Jena, Germany
4 Institute for Biochemistry I, University of Cologne, Joseph-Stelzmann-Str. 52, 50931
Cologne, Germany
5 Berlin Center for Genomics in Biodiversity Research, Königin-Luise-Str. 1-3, 14195
Berlin, Germany
6 Universität Bielefeld, CeBiTec, 33594 Bielefeld, Germany
§Corresponding author
Uwe John
[email protected] Manuscript 1 32
Abstract
Background
The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP) toxins, which are known only from cyanobacteria and dinoflagellates.
While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive.
Results
We constructed and analysed an expressed sequence tag (EST) library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences,
192 genes were differentially expressed between toxic and non-toxic strains.
Conclusions
Based on this study and on the lack of identified PSP synthesis genes in the two existent
Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins. Manuscript 1 33
Background
Alexandrium minutum is a bloom-forming toxic dinoflagellate typically capable of producing paralytic shellfish poisoning (PSP) toxins. Occurring from northern Europe and the Mediterranean to Asia, Australia and New Zealand, A. minutum poses a widespread threat to seafood production and consumer health (for a review of biogeography and species boundaries see Lilly et al., 2005). PSP is potentially fatal to both humans (García et al., 2004) and marine fauna, particularly vertebrates (Geraci et al., 1989; Trainer & Baden, 1999); human intoxications typically occur after ingestion of suspension-feeding bivalve molluscs.
PSP toxins are tetrahydropurine neurotoxins, which specifically bind to voltage-gated sodium channels of nerves and muscle cells. The most well known representative, saxitoxin, is probably also the parent compound (Sako et al., 2001), of which more than
20 naturally occurring derivatives are known, including the gonyautoxins produced by
A. minutum (reviewed in Krock et al., 2007). Several physiological or ecological functions of PSP toxins have been considered (reviewed in Cembella, 1998), among others chemical defence (Cembella, 1998), nitrogen storage (Cembella, 1998), activity as pheromones (Wyatt & Jenkinson, 1997), and influences on the dinoflagellate- associated bacteria in the phycosphere (Jasti et al., 2005). Several experimental studies link PSP toxicity in dinoflagellates to deterring effects on some species of copepod grazers (Turner & Tester, 1997; Selander et al., 2006), which can lead to a redirection of grazing pressure onto non-toxic phytoplankton species competing for the same nutrient resources (Guisande et al., 2002), but the interactions and deterrence responses are highly species-specific and not universal. Manuscript 1 34
In addition to these effects, but independent of PSP toxin content, several Alexandrium species including A. minutum were shown to produce lytic substances which affect both competing algae and unicellular predators (Tillmann & John, 2002; Tillmann et al.,
2008). Both these factors potentially contribute to the formation and maintenance of A. minutum blooms.
Analytical and physical-chemical methods can resolve structures and provide basic biosynthetic pathways to phycotoxins, but provide little direct evidence on specific biosynthetic enzymes and regulatory functions (Cembella & John, 2006). Thus, a combined approach linked to genomics and proteomics is required to fully describe toxin biosynthesis and regulation. In the case of PSP toxins in dinoflagellates, however, this approach is rather challenging, as both the physiological regulation and evolutionary origin of PSP toxins remain enigmatic.
Apart from marine dinoflagellates, comprising several species of Alexandrium and a single species Pyrodinium bahamense of a closely related genus, plus Gymnodinium catenatum, which belongs to a very distantly related phylogenetic group (see
Saldarriaga et al., 2001; Levy et al., 2007), the only confirmed PSP toxin producers are predominantly freshwater cyanobacteria, including members of the genera
Cylindrospermopsis, Anabaena, Lyngbya and Aphanizomenon. This paraphyletic distribution was confirmed by several studies demonstrating that removal of bacteria from Alexandrium cultures did not eliminate toxin production (e.g. Lu et al., 2000; Hold et al., 2001; Uribe & Espejo, 2003). Along with the lack of conclusive evidence that toxigenesis could be definitively attributed to endosymbiotic bacteria within dinoflagellates, this supports the conclusion that the dinoflagellate genome is responsible for PSP toxin production. As the inheritance of toxin composition follows a
Mendelian segregation pattern (Ishida et al., 1998), at least the interconverting enzymes Manuscript 1 35 are almost certainly encoded by nuclear genes. Since the early investigations into the biosynthetic pathway of PSP toxins (Shimizu et al., 1984), the standing hypothesis has been that this pathway is the same in all PSP toxin-producing organisms, and that the corresponding genes should be homologous. In one of the most-cited papers on his model of the PSP toxin synthesis pathway, Shimizu (Shimizu, 1996) suggested that the unusual distribution of the ability to produce PSP toxins might be explained by a rare event of horizontal gene transfer from bacteria to dinoflagellates.
A cyanobacterial PSP toxin gene cluster (sxt) has recently been discovered in
Cylindrospermopsis raciborskii T3 (Kellmann et al., 2008b). This cluster contains >35 kb of sequence coding for 26 proteins; PSP toxin synthesis is reported to be initiated with arginine, S-adenosylmethionine and acetate by a new type of polyketide synthase.
Similar clusters were found in other PSP toxin-producing cyanobacterial species
(Mihali et al., 2009; Stucken et al., 2010), and further studies suggested that at least parts of this cluster have been frequently laterally transferred between different cyanobacterial genera (Kellmann et al., 2008a). Nevertheless conclusive evidence of the homology of PSP toxin synthesis in dinoflagellates and prokaryotes is pending.
Attempts to find Alexandrium genes associated with PSP toxin production have remained inconclusive. While the physiological and circadian time-frame of PSP toxin production has been elucidated in A. fundyense (Taroncher-Oldenburg et al., 1997), further experiments based on this information have failed to identify candidate genes directly linked to PSP toxins (Taroncher-Oldenburg & Anderson, 2000). Subtractive hybridization of cDNA was used to identify toxin-associated genetic differences between toxic and non-toxic subclones of one Alexandrium tamarense parental strain
(Cho et al., 2008). The differential gene fragments, however, did not seem to be directly related to toxins, as shown by polymorphism analysis of other subclones. Manuscript 1 36
Genomic studies in dinoflagellates have long been hampered by their huge genomes, particularly of free-living species – up to 200 pg nuclear DNA cell-1 in Prorocentrum micans, which corresponds to roughly 75 times the DNA content of the human genome
(LaJeunesse et al., 2005) - and their unusual nuclear composition and organisation
(reviewed by Moreno Díaz de la Espina et al., 2005). Although most dinoflagellates are functionally haploid in the vegetative stage, the nucleus may contain up to 270 chromosomes (Moreno Díaz de la Espina et al., 2005). The dinoflagellate nucleus typically lacks histones and nucleosomes and instead contains low abundances of basic histone-like proteins. The chromosomes are permanently condensed, and up to 70% of the nucleotides contain modified or rare bases. This unusual organisation was further illustrated in a low-redundancy sequence survey of the Heterocapsa triquetra genome
(McEwan et al., 2008); about 90% of the examined sequences were apparently random, non-repetitive DNA, whereas the highest number of recognizable sequences consisted of repeats, transposons or virus-specific protein sequences. These features pose major difficulties in directly studying the gene content of dinoflagellates, and while some sequencing initiatives are underway, no completely sequenced genome is yet available.
Most of the complexities associated with examining the dinoflagellate genomes can be circumvented by conducting genomic studies at the transcriptomic level (Rudd, 2003).
In the last few years, a number of expressed sequence tag (EST) studies has been published (e.g. Tanikawa et al., 2004; Hackett et al., 2005; Lidie et al., 2005; Patron et al., 2005; Jaeckisch et al., 2008; Uribe et al., 2008), many of which led to further microarray-based studies of gene expression (e.g. Okamoto & Hastings, 2003a; Van
Dolah et al., 2007). In dinoflagellate species producing polyketide toxins of the spirolide and brevetoxin groups, search strategies based on EST- or cDNA-libraries were successful in identifying a range of polyketide synthases (PKS) (Jaeckisch et al.,
2008; Monroe & Van Dolah, 2008). These studies made use of the high degree of Manuscript 1 37 sequence conservation in several PKS domains (reviewed in John et al., 2008), but which of these genes are responsible for production of the corresponding toxins has not yet been established.
As well as being a member of the most well-studied dinoflagellate genus of PSP toxin- producers, for which a wealth of physiological and biosynthetic information on toxin production is available, A. minutum is an appropriate model for toxin production studies because of its simple toxin profile. Most A. minutum strains produce mainly or exclusively gonyautoxins and ab initio both toxic and non-toxic strains are available for comparative studies (Tillmann & John, 2002; Touzet et al., 2007). We constructed a normalised EST library of A. minutum to search for PSP toxin-related genes in this dinoflagellate. To search for toxin-related or toxicity-influencing genes not readily apparent from EST library annotation, we used a microarray approach to compare the transcriptomes of toxic and non-toxic A. minutum clones and thus identified differences in gene expression potentially linked to toxin synthesis and/or regulation.
Methods
General culture conditions for Alexandrium minutum
Unless noted differently, A. minutum cultures (origin: Gulf of Trieste, Italy) were grown
-1 - at 20°C in modified K-medium (Keller et al., 1987) containing 440 µmol L NO3 , 36
-1 + -1 3- -1 2- -1 µmol L NH4 , 25 µmol L PO4 , 10 nmol L SeO3 , 1000 µmol L Trizma-Base (pH
8.3), K trace-metal solution and f/2 vitamin solution (Guillard & Ryther, 1962; Guillard,
1975). Illumination was provided from daylight fluorescent lamps at a photon flux density (PFD) of 200 µmol m-2 s-1 on a 16:8-hour light:dark photocycle. All experimental (but not stock) cultures were grown under antibiotic treatment (50 µg mL-1 ampicillin, 33 µg mL-1 gentamicin, 10 µg mL-1 ciprofloxacin, 1.13 µg mL-1 Manuscript 1 38 chloramphenicol and 0.025 µg mL-1 streptomycin sulphate) using sterile handling techniques to minimize bacterial influence. To avoid any bias introduced by the antibiotics, treatment was stopped at inoculation of the cultures for toxin content and gene expression experiments, but aseptic handling techniques were maintained.
Culture and harvest of A. minutum for EST library construction
A. minutum clone AL3T was grown in 800 mL culture bottles for EST library construction. In order to include genes expressed under different physiological conditions, several alternative treatments were applied. Standard condition cultures were grown as detailed above. Two standard condition cultures were subjected to shock treatments (complete darkness or 5°C) for 24 h prior to harvesting. Cultures treated in high salinity (medium supplemented with 15 g L-1 NaCl) and low salinity (medium prepared with 1/3 aged seawater and 2/3 deionised water) were pre- conditioned in these media for ca. 40 cell cycles (about 40 days, over several sub-culturing transfers) before inoculation of the final cultures. To achieve N-limitation at a reasonably high cell
- -1 + concentration, NO3 , but not 36 µmol L NH4 , was omitted from the culture medium.
For P-limitation, cultures were grown in medium to which no phosphate was added.
Light limitation of growth was attained under a PFD of 60 µmol m-2 s-1, and for high and low temperature conditions, cultures were grown at 31.5°C and 13°C, respectively.
The cultures were harvested at 7 time points through the photocycle to capture genes expressed at specific time points, and at different stages of the growth curve.
Cells were harvested by filtration upon an 8 µm pore-sized filters and rinsed with filter- sterilized seawater. Cells were immediately transferred into TriReagent (Sigma-Aldrich,
Steinheim, Germany), or filters were quick-frozen in liquid nitrogen for later processing. Manuscript 1 39
EST library construction and automated annotation
RNA was extracted using the Sigma TriReagent protocol, following cell lysis by 10 min incubation at 60°C in TriReagent, aided by repeated vortexing with glass beads included in the sample tube. The resulting RNA pellet was dissolved in 100 µL RNAse-free water (Qiagen, Hilden, Germany). RNA cleanup including on-column DNA digestion
(27.3 u DNase per sample) followed the protocol supplied with the Qiagen RNeasy kit.
RNA was eluted with 40 µL RNase-free water (Qiagen). When necessary, an additional cleanup and concentration step was applied using Qiagen MinElute or Microcon
Ultracel YM-30 columns. RNA purity and quantity were determined with a NanoDrop spectrophotometer (PeqLab, Erlangen, Germany), and integrity and absence of DNA contamination was assessed with a Bioanalyzer (Agilent Technologies, Böblingen,
Germany).
Total RNA from harvested cells was pooled as equal amounts from each treatment for construction of a normalised cDNA library. The library was constructed and transformed into electrocompetent Escherichia coli cells by Vertis Biotechnologie AG
(Freising-Weihenstephan, Germany).
Colonies were picked, and the DNA was extracted by magnetic beads on a robot platform (Qiagen, Hilden, Germany). Plasmid inserts were sequenced from both sides using Big Dye Chemistry (Applied Biosystems, Darmstadt, Germany) and separated on an ABI Prism 3700xl sequencing (Applied Biosystems) platform. Resulting high quality sequence reads (15,703) were clustered with a sequential assembly using decreasing identity thresholds (gap assembler) to avoid misassemblies due to polyA tails. Contigs were searched for potential alternative splicing by checking alignments of reads in clusters for the presence of gaps in at least one read compared to the consensus sequence of the cluster. Alternative splicing was assumed if the following criteria were Manuscript 1 40 fulfilled: 1) the manually inspected difference between the transcripts was at least two bases, and 2) no other polymorphisms were present between the alternative transcripts, thereby indicating transcription from the same locus.
Contig sequences were loaded into SAMS (Sequence Analysis and Management
System, Center for Biotechnology, Bielefeld University) for automated annotation based on BLAST comparisons against KEGG, KOG, SwissProt, InterPro and the
Genbank nt and nr databases. Sequences were subject to open reading frame (ORF) prediction according to (Min et al., 2005), and predicted ORFs were submitted to Pfam,
TMHMM-2.0 and SignalP 3.0. To further assist manual annotation, all contigs were analyzed for phylogenetic association to SwissProt sequences using PhyloGena
(Hanekamp et al., 2007). Annotations of 192 sequences identified from the microarray results as differentially expressed in toxic and non-toxic strains were checked manually.
In-silico search for known sxt-related genes
The assembled A. minutum EST library was screened for contigs showing similarities to any of the publicly available (as of 13.1.2009) sxt-related cyanobacterial sequences in the NCBI protein database using translated protein – nucleotide (blastx) BLAST
(Altschul et al., 1997). Contigs and cyanobacterial sequences producing significant hits
(e-value < 0.001) were subjected to a PhyloGena analysis in “top10select” mode to identify the most similar SwissProt sequences and to obtain the most likely protein translation of the EST contig sequence. BLAST-identified pairs of sxt proteins and translated A. minutum contigs were combined with the full-length versions of their respective SwissProt hits, and, in the cases of Amin_93i12r and Amin_73a05f, additionally with the 20 most similar nr sequences as identified by BLAST. These sequence sets were used for phylogenetic analysis following the method detailed in
(John et al., 2008) with modifications. Briefly, alignments were generated from kalign, Manuscript 1 41 and alignment blocks out of the span of both the A. minutum and the sxt-related sequence were manually deleted with the CLC sequence viewer version 6.0.1 (Knudsen et al.). Phylogenetic trees were calculated with Phyml_3.0.1 (Guindon & Gascuel,
2003) using the subtree pruning and regrafting (SPR) method with a BioNJ starting tree, and the LG model of amino-acid substitution (Le & Gascuel, 2008) with gamma distribution parameters estimated from the data with four discretised substitution rate classes, the middle of which was estimated as the median. The same settings were used for a 100-replicate Bootstrap analysis.
Microarray design
Oligonucleotide probes covering both forward and reverse reading directions were designed in collaboration with CeBiTec, Bielefeld University. After a first test hybridisation of a microarray containing 22,264 oligos (2-14 per contig, mean 5.2), the probe set was reduced to the 8,609 best-performing oligos. Following a test for congruence with the contig ORF direction as determined by either manual annotation or a combination of PolyA-Tail identification and the direction of the best SwissProt hit, the best probe was chosen according to performance in triplicate hybridization with standard-strain (AL3T) RNA from 9 different physiological conditions. For some contigs without available manual annotation which could not be automatically assigned an ORF direction, two probes targeting both possible mRNA orientations were retained; these are marked as correct or false during manual annotation.
Toxin analysis
PSP toxins were extracted following a previously described method (Krock et al.,
2007). After centrifugation (3,000 x g, 4°C), pellets were suspended in 0.03 N acetic acid and homogenized in FastPrep tubes (Thermo Savant, Illkirch, France) containing
0.9 g of lysing matrix D by reciprocal shaking in a Bio101 FastPrep instrument (Thermo Manuscript 1 42
Savant) at maximum speed (6.5 m s-1) for 45 s. Cell debris was removed by centrifugation at 16,100 x g at 4°C for 15 min. The supernatant (400 µL) was filtered through a 0.45 mm pore-size spin-filter (Millipore Ultrafree, Eschborn, Germany) by centrifugation for 30 s at 800 x g.
Analytical Reagents
Water was deionised and purified (Milli-Q, Millipore, Eschborn, Germany) to 18
MΩ cm-1 quality or better. Formic acid (90%, p.a.), acetic acid (p.a.) and ammonium formate (p.a.) were purchased from Merck (Darmstadt, Germany), nitric acid (p.a.) and phosphoric acid (p.a.) were from AppliChem (Darmstadt, Germany), periodic acid, 1- heptanesulphonic acid, 1-octanesulphonic acid and diammonium hydrogenphosphate were from Sigma (Deisenhofen, Germany). The solvents, methanol, tetrahydrofurane
(THF) and acetonitrile, were high performance liquid chromatography (HPLC) grade
(Merck, Darmstadt, Germany).
Standard solutions of PSP toxins (saxitoxin, STX; neosaxitoxin, NEO; decarbamoyl saxitoxin, dcSTX; gonyautoxins 1&4, GTX1/GTX4; gonyautoxins 2&3 GTX2/GTX3; decarbamoyl gonyautoxins 2&3, dcGTX2/dcGTX3; and B1) were purchased from the
Certified Reference Material Programme of the Institute for Marine Biosciences,
National Research Council, Halifax, NS, Canada.
Analytical Methods
Liquid Chromatography with Fluorescence Detection (LC-FD)
The LC-FD analysis was carried out as previously described in detail (Krock et al.,
2007). Briefly, PSP toxins were separated by ion-pair chromatography on a ODS reverse-phase analytical column and detected fluorometrically after post-column derivatization. PSP toxins were resolved with two eluants. The first eluant for gonyautoxins and N-sulfocarbamoyl toxins consisted of 6 mM 1-octanesulphonic acid Manuscript 1 43 and 6 mM 1-heptanesulfonic acid in 40 mM ammonium phosphate, adjusted to pH 7.0 with dilute phosphoric acid. Components of the saxitoxin group were separated by
0.75% THF and 13 mM 1-octanesulphonic acid in 50 mM phosphoric acid, adjusted to pH 6.9 with ammonium hydroxide, and 15% (v/v) acetonitrile and 1.5% THF. The flow rate was 1 mL min-1 with the following gradient: initial condition: 100% eluant A and isocratic elution until 15 min, gradient elution to 100% eluant B until 16 min, followed by isocratic elution with 100% eluant B until 35 min.
Post-column derivatization was performed with 10 mM of periodic acid in 550 mM ammonium hydroxide at a flow rate of 0.4 mL min-1 in a reaction coil set at 50°C.
Subsequently, the eluate was continuously acidified with 0.75 N nitric acid at a flow rate of 0.4 mL min-1. Toxins were detected by a dual monochromator fluorescence detectoU ȜH[ QP ȜHP QP 363 WR[LQ FRQFHQWUDWLRQV ZHUH GHWHUPLQHG E\ external calibration.
Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS)
In order to confirm the LC-FD results using an independent method, mass spectral experiments were performed on an ABI-SCIEX-4000 Q Trap (Applied Biosystems,
Darmstadt, Germany), triple quadrupole mass spectrometer equipped with a
TurboSpray® interface coupled to an Agilent (Waldbronn, Germany) model 1100 LC.
The LC equipment included a solvent reservoir, on-line degasser (G1379A), binary pump (G1311A), refrigerated autosampler (G1329A/G1330B), and temperature- controlled column oven (G1316A).
Toxin separation (5 µL of sample injected) for mass spectrometric analyses was performed by a hydrophilic interaction liquid ion-chromatography (HILIC) method
(Diener et al., 2007) with slight modifications. The analytical column (150 x 4.6 mm) was packed with 5 µm ZIC-HILIC, 200 Å, (SeQuant, Haltern, Germany) and Manuscript 1 44 maintained at 35°C. A pre-column with the same packing material was also used. The flow rate was 0.7 mL min-1 and gradient elution was performed with two eluants. Eluant
A was 2 mM formic acid and 5 mM ammonium formate in 20% water and 80% acetonitrile; eluant B was 10 mM formic acid and 10 mM ammonium formate in water.
The gradient was as follows: 20 min column equilibration with 20% eluant B, linear gradient to 35% B until 5 min, then linear gradient to 40% B until 10 min, then linear gradient to 45% eluant B until 20 min, followed by isocratic elution with 45% eluant B until 24 min and finally return to initial 20% eluant B until 25 min.
Multiple reaction monitoring (MRM) experiments were carried out in positive ion mode by selecting the following transitions (precursor ion > fragment ion): m/z 412>332 and m/z 412>314 (for GTX1/GTX4 and C3/C4), m/z 396>316 and m/z 396>298 (for
GTX2/GTX3, C1/C2 and B2), m/z 380>300 and m/z 380>282 (for B1), m/z 353>273
(for dcGTX2/dcGTX3), m/z 369>289 (for dcGTX1/dcGTX4), m/z 300>282 and m/z
300>204 (for STX), m/z 316>298 and m/z 316>196 (for NEO), m/z 257>196 and m/z
257>156 (for dcSTX) and m/z 273>255 (for dcNEO). Dwell times of 50–150 ms were used for each transition with the following source parameters: curtain gas: 30 psi, temperature: 650°C, ion-spray voltage: 5000 V, gas 1 and 2: 70 psi, interface heater: on, collision gas: high, declustering potential: 66 V, entrance potential 10 V, collision energy: 30 V, and collision cell exit potential: 12 V.
Determination of harvesting time for gene expression analysis
To determine the optimal point of the light-dark cycle at which putative toxin genes are most likely to be expressed, triplicate A. minutum AL3T cultures were grown in 5 L glass flasks with constant gentle aeration under controlled standard conditions, as previously described. Cultures were sampled for cell counts and toxin analysis at the onset of dark phase, at the onset of light phase, and at 2-h intervals until the beginning Manuscript 1 45 of the next dark phase, with a sterile tube-sampling system (John et al., 2000; Eschbach et al., 2005). Duplicate samples for toxin analysis (125 – 200 mL of culture depending on harvesting time) were harvested by centrifugation for toxin analysis by LC-FD.
Inter-clonal comparisons of gene expression
Gene expression was compared between toxigenic (AL3T and AL9T) and naturally non-toxic (AL1T) A. minutum clonal strains originating from the same geographical population in the Gulf of Trieste, Italy (isolated by A. Beran, see Montresor et al.,
2004). Bacteria-reduced triplicate cultures were grown under the control conditions as stated above. Culture growth was monitored by daily manual cell counts of samples fixed in Lugol’s iodine solution.
The toxin content of duplicate samples containing at least 2 x 106 AL3T and AL9T cells and non-duplicate samples containing at least 4 x 106 AL1T cells were analyzed by LC-
FD. To confirm the toxin identification of specific PSP toxin analogues by LC-MS, samples of additional parallel cultures were combined into pellets containing 8.5 x 106 to 1.1 x 107 cells.
Samples for RNA extraction were taken during exponential growth phase, at 10-11 h
(Sampling Time 1, ST1) and at 6-7 h (Sampling Time 2, ST2) after onset of light phase.
Total RNA (500 ng sample-1) was extracted as described herein, then amplified and labelled with a Low RNA Input Linear Amplification kit (Agilent, Waldbronn,
Germany). The Agilent Low RNA Input Linear Amplification Kit protocol was followed for synthesis of Cy3- and Cy5-labelled cRNA and microarray hybridisation.
Microarrays were scanned on an Agilent G2565AA scanner, and raw data were extracted with the Agilent Feature Extraction Software version 9.1.3.1 (FE). Array quality was monitored with the Agilent QC Tool (v1.0) with the metric set
GE2_QCMT_Feb07. Manuscript 1 46
Pre-processed data were subjected to SAM (Significance Analysis of Microarrays,
Tusher et al., 2001) as implemented in MeV 4.0 (Saeed et al., 2006), and SAM-based q- values (Storey, 2003) were calculated. Probes with a q-value of <1% were considered to indicate differential expression of the corresponding genes if the mean fold change of the sample triplicate was at least 1.5. To minimise the influence of physiological differences between the strains that were not related to the capacity for toxin production, only genes identified as significantly higher or significantly less expressed in both toxic strains at both sampling time-points were designated as “higher expressed” or “less expressed” in toxic strains.
Samples were clustered by Hierarchical Cluster Analysis (HCL) support trees as implemented in MeV 4.2. Trees calculated with different distance measures (Euclidean,
Manhattan and Covariance values) and different distance metrics (average, complete and single linkage) were compared. Node confidence was tested by 1000 Bootstrap replicates.
To test reliability of the microarray, expression levels of six genes were evaluated by qPCR. Four of these genes had been identified as significantly higher expressed in the toxic strains, the other two as significantly higher expressed in the non-toxic strain. The qPCR was carried out as described (Krell et al., 2007) with the lepidopteran genes MA and NSP as an artificial internal reference. Primers for qPCR were designed with
PrimerExpress 3.0 (Applied Biosystems, Darmstadt, Germany) and synthesized by
Eurofins MWG Operon (Ebersberg, Germany). Standard curves using cDNA plasmids corresponding to target sequence ESTs were plotted to test the primer pairs for consistent efficiency at different concentrations. These plasmids were amplified by
M13-primed PCR; qPCR primers were tested on dilution series of plasmid PCR products spanning at least 8 orders of magnitude. The qPCR reaction was based on the Manuscript 1 47
PowerSybrGreen PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions, using a 7000 Real-Time PCR System (Applied
Biosystems).
Prior to cDNA synthesis the samples were spiked with the artificial internal control
RNAs MA (1 ng reaction-1) and NSP (1 pg reaction-1). Equal amounts of RNA from all strains were processed in parallel. All qPCR reactions based upon the same primer-set were run on the same plate, and reaction efficiencies were compared with MA and NSP- specific primers. To test for contamination with genomic DNA, negative controls consisted of full reactions in which cDNA was exchanged for RNA aliquots of all samples. Thresholds were determined manually for each primer set. Relative expression levels were recorded as the cycle threshold value (Ct).
PCR
To test for the presence of the cyanobacterial toxin gene cluster in A. minutum, amplification of the cyanobacterial genes from genomic DNA isolated from AL3T cultures grown under antibiotic treatment was attempted. The 14 primer pairs designed against the PSP toxin ORF in the Raphidiopsis brookii genome (K. Stucken et al., unpublished data; Additional file 1) were tested. A primer pair targeting the D1/D2
(Scholin et al., 1995) region of the large ribosomal subunit was used as positive control, primers against the bacterial 16S ribosomal subunit controlled for bacterial contamination of the DNA.
Reactions were scaled to 30 µL and contained 20 ng genomic DNA, 0.2 mM forward and reverse primers, 0.1mM dNTPs, 0.6 units of U of HotMaster Taq DNA polymerase
(Eppendorf) and HotMasterTaq buffer 1X (Eppendorf). Gradient PCR was employed to try each primer at 6 different annealing temperatures spanning a range of approximately
9°C. As the primers were of different melting temperatures, three different temperature Manuscript 1 48 spans between 46 and 58°C were used. Cycling conditions were based on those optimal for these primers (Additional file 1): initial denaturation at 94°C for 5 min, 20 cycles with denaturation 94°C (20 s), annealing for 45 s, extension at 70°C (30 s), and a final extension step of 10 min at 70°C. PCR products were visualized after agarose gel electrophoresis.
Results
Characterisation of the EST Library
The cDNA fragments sequenced from both sides yielded 15,703 read sequences clustered into 4,320 contigs. Of these contigs, 3,112 (72%) combined the forward- and reverse reads of at least one bacterial clone, and thus can be considered full-length insert sequences, but might not represent the total length of the corresponding mRNA. Among contigs, 1,998 contained reads from more than one cDNA insert. Contig length varied between 100 and 2185 bp, with a mean of 791±240 bp (mean ± standard deviation). The mean GC content of contigs was 56.10 ± 4.67% (Table 1).
The examination of alignments of reads in the clusters for potential alternative splicing identified different splice variants in ca. 9% of the contigs containing reads from more than 1 cDNA insert, or ca. 3% of all genes examined.
Checking all contigs for evidence of transsplicing revealed that the complete dinoflagellate-specific spliced leader sequence (SL),
DCCGTAGCCATTTTGGCTCAAG (D =U, A, or G) (Lidie & Van Dolah, 2007;
Zhang et al., 2007) was present in 204 contigs. Of these SLs, 37 (18%) started with A,
10 (5%) with G and 157 (77%) with U. In addition, we identified 35 contigs containing incomplete SL sequences. In 23 of these, the sequence was lacking 1-4 nucleotides at the 5’-end of the contig, while 23 SLs differed in 1-3 nucleotides within the SL Manuscript 1 49 sequence. Of the SL-containing contigs, 193 contained an additional 5’polyA-region and thus are likely to contain the complete ORF (see Additional file 2).
Automated annotation with the SAMS platform yielded annotations for 1,200 contigs
(28%), whereas 1,969 contigs (46%) did not produce any BLAST hit below tool cut-off.
Another 1,151 contigs (27%) produced no hits that met the criteria for automated annotation. Automated classification to KOG categories based on the SAMS “BLAST
2x vs. KOG” output (cut-off e-value 10-4) resulted in assignment of 1005 contigs (23%) to functions other than “General function prediction only” or “Function unknown”
(Fig.1).
A BLAST survey of the A. minutum library against all available dinoflagellate EST libraries available in GenBank (as of 28.11.2007) plus a new A. ostenfeldii EST library
(N. Jaeckisch, unpublished observations) detected similar sequences for 43% of the contigs at a moderately stringent cut-off value of e = 1 x 10-10 (Table 2).
In-silico search for sequences related to the cyanobacterial PSP toxin gene cluster
A BLAST search of the EST library against the cyanobacterial PSP toxin genes resulted in 14 hits at e-value <0.001. In 12 of 14 maximum likelihood phylogenies calculated from the BLAST-identified pairs of sxt proteins and translated A. minutum contigs, and their closest matches identified from the SwissProt database, the A. minutum and the cyanobacterial sequences clustered in different clades. Furthermore, these clades were separated by long branches, well-supported nodes with high bootstrap support, or both
(see Additional file 3: Phyml-based likelihood trees with bootstrap support values). In the case of Amin_93i12r and Amin_73a05f, the phylogenies based on SwissProt hits were not that clear (Additional file 3 M.1 and N.1). This led to a further test for orthology of Amin_93i12r and Amin_73a05f with the cyanobacterial PSP toxin genes by including the results of a BLAST search against the nr database. In the case of Manuscript 1 50
Amin_93i12r, the cyanobacterial gene in question was identified in this BLAST search as hit number 64; the BLAST result list of Amin_73a05f did not include its potential cyanobacterial counterpart within the best 100 hits. When we repeated calculation of the phylogenies for Amin_93i12r and Amin_73a05f with the best 20 BLAST vs. nr hits included in addition to the SwissProt database hits, the Alexandrium sequences did not cluster in the vicinity of the cyanobacterial PSP toxin genes (Additional file 3 M.2 and
N.2).
Toxin production over the light-dark cycle
Under our standard growth conditions, the toxin-producing strain AL3T exhibited a growth rate (1.01 cell divisions day-1; I. Yang, unpublished data) that approximated the total length of the light:dark period, which suggested that these cultures might have a naturally circadian-phased cell cycle. In the triplicate cultures examined, however, both cell number and culture toxin content increased over the entire light phase. The toxin content per cell stayed roughly the same during the whole day, while cell numbers doubled from 3,271 ± 296 to 7,167 ± 482 cells mL-1 within 24 h. The toxin content of the culture increased from 1.38 ± 0.16 ng µL-1 to 3.24 ± 0.21 ng µL-1 (Fig.2)
Strain-specific toxin content
A. minutum PSP toxin concentration as measured by LC-FD was strongly clone- dependent. No PSP toxins were found in AL1T, whereas the toxin profiles of AL3T and
AL9T were virtually identical, although toxin cell quotas differed by an order of magnitude (Table 3). The toxin profiles as confirmed by LC-MS/MS (Fig. 3) in the extracted ion chromatograms clearly indicate the presence of the transitions m/z 396 >
298 for GTX3 and m/z 412 > 314 for GTX4. No signals were obtained from any other ion traces, indicating that no known PSP toxins other than GTX3 and GTX4 were present in the samples. Manuscript 1 51
Gene expression
Gene expression differences between toxic and non-toxic isolates were identified by microarray experiments comparing the two toxic strains AL9T and AL3T to the non- toxic AL1T. With SAM at a gene-specific FDR cut-off of q=1% and a fold-change cut- off of 1.5, 145 contigs were identified as higher expressed in both toxic strains at both time points tested, whereas 47 genes were significantly higher expressed in the non- toxic strain (see Additional file 2 – List of differentially expressed genes). HCL support trees in which samples were clustered according to similarity of gene expression grouped the samples according to their clonal designations. Of the nine tested different combinations of clustering parameters, only the tree based on the Euclidean distance measure combined with complete linkage separated the midday and afternoon triplicates of the AL9T vs. AL1T microarrays. In all other analysis, samples did not cluster according to harvesting time.
Of the 145 genes identified as higher expressed in the toxic strains at both time points,
49 could be manually annotated based on BLAST-based automated annotation, domain comparison using Pfam and phylogenetic reconstruction with the PhyloGena program
(Hanekamp et al., 2007). Putative functions could be assigned to 31 of these genes
(Table 4).
The group of 47 genes higher expressed in the non-toxic strain AL1T contained 8 genes for which a putative function could be identified (Table 4).
For 8 of the contigs higher expressed in the toxic strains, the signals of the non-toxic strain were identified as not “well above background” by the microarray data extraction software on at least 8 of the 12 arrays, and on at least one array for each strain-time- point combination. We consider these as either not expressed in the non-toxic strain
AL1T or considerably different on the sequence level, which might imply functional Manuscript 1 52 differences. This group contains two putative helicases and six hypothetical proteins for which no annotation could be found. For one of these, Amin_66c10r, we found a homologous sequence in the published A. tamarense library (Hackett et al., 2005), but in none of the libraries of non-PSP toxin-producing dinoflagellates. Another five sequences examined did not produce significant BLAST hits against any other dinoflagellate library (see Additional file 2).
The analysis of relative mRNA levels of 6 genes re-examined by qPCR, as shown in
Fig. 4, indicated that microarray and qPCR data agreed qualitatively, although most of the fold changes calculated from qPCR results were substantially higher than those identified by microarray.
Discussion
EST Library
The EST library presented here includes sequences from a variety of physiological states induced by different treatments and sampling time points. We aimed for a high diversity of transcripts ideally representing an overview over the complete transcriptome of this species, by combining samples from control, cold-shocked, darkness-shocked, high- and low-salinity stressed, nitrogen- and phosphorous-starved as well as heat- and cold-stressed A. minutum cultures and by normalization of the cDNA library. The 15,703 read sequences, which originated from 9,485 cDNA clones, clustered into only 4,320 contigs or putative genes. We estimated that our contigs account for approximately 80% of the potential genes within this particular cDNA library, based upon the rarefaction curve analysis (Additional file 4). Nevertheless, we recognize that not 80% of the potential gene candidates from the A. minutum genome are included in the library. Based on the experience of diverse genome projects, the total Manuscript 1 53 number of gene candidates is probably two or three times higher. In any case, contig numbers for the A. minutum library are substantially lower than those found for EST libraries of the congeneric A. tamarense (6,723 unique sequences from 11,171 reads, normalised library) (Hackett et al., 2005) and A. catenella (6,496 “unigenes” from
10,850 ESTs, library not normalised) (Uribe et al., 2008). The latter two EST libraries were both obtained from cultures grown only under control conditions, and hence presumably do not reflect stress, cell cycle or different culture stage effects on gene expression. The relationship of unique genes within the EST library for A. minutum is reminiscent of that of the free-living marine dinoflagellate Heterocapsa triquetra
(Patron et al., 2005), for which the EST library was based on samples taken throughout the photocycle, but contained only 2,022 unique clusters out of 9,309 ESTs.
A BLAST-search of the A. minutum contigs against available dinoflagellate EST libraries found at least weakly similar sequences for about half of the contigs, which is mainly due to the availability of EST libraries for A. tamarense (Hackett et al., 2005),
A. catenella (Uribe et al., 2008) and A. ostenfeldii (N. Jaeckisch et al, unpublished observations) (Table 2). The low novelty rate implied by these similarities, however, is not reflected in the annotation results; automated annotation was possible for 28% of the contigs. The attribution of these contigs among functional categories (depicted in Fig.1) is likely to reflect to a large extent the sequence conservation in the respective groups, and not necessarily the true distribution of gene functions. Studies examining the function of dinoflagellate genes are still rare, and as they are ecologically, physiologically and genetically very distinct from their better-studied relatives, the apicomplexa (Fast et al., 2001), annotation of dinoflagellate genes remains notoriously difficult (see John et al., 2004). Manuscript 1 54
A recent study (Bachvaroff & Place, 2008) suggests that gene expression in dinoflagellates reflects genomic organisation. Most highly expressed genes seem to be in tandem arrays of slightly different gene copies, with few or no introns, short intergenic spacers, and are mostly spliced-leader trans-spliced (Lidie & Van Dolah,
2007; Zhang et al., 2007). We found evidence for the existence of such gene families for 8 of 9 sequences tested; in addition to intra-specific differences among strains, there was considerable intra-strain variability on both the transcriptomic and genomic levels, with variation in the cDNA within a strain, comprising both synonymous and non- synonymous differences. This implies that microarray-measured differences in gene expression might be due to either differences in the frequencies of highly probe- complementary vs. less probe-complementary gene family members, or to the simultaneous up- or down-regulation of whole gene families.
In contrast to these multi-copy genes, moderately expressed genes appear to be single copy and exhibit a higher intron density; trans-splicing seems to occur less often
(Bachvaroff & Place, 2008). Furthermore, we identified alternative splice variants in 9% of the genes containing reads from more than one cDNA, adding to the evidence that more conventional eukaryotic introns and splicing phenomena exist in dinoflagellates
(Alverca et al., 2006).
Based on our data and with reference to previous studies we cannot, however, exclude that in rare cases identical copies with different splicing patterns may exist. Yet since splicing accuracy and efficiency depends on the underlying sequence, this scenario is highly unlikely. In any case, to our knowledge, our work herein is the first report on the frequency of alternative splicing in dinoflagellates. Manuscript 1 55
In-silico search for candidate saxitoxin-related genes
The known information on PSP toxin-related proteins in dinoflagellates is limited to enzymes involved in the interconversion of PSP toxins, which have been characterised on the protein level. An oxidase capable of transforming GTX 3 to GTX 4 has been found in crude extracts of A. tamarense cells (Oshima, 1995). In the dinoflagellate
Gymnodinium catenatum the same research group also reported an N-sulfotransferase forming C2 from GTX3 in a reaction mix containing adenosine 3’-phosphate 5’- phosphosulfate. Nevertheless, this enzyme was present in both toxic and non-toxic isolates of this species. A similar enzyme was also found in a crude enzyme extract of
A. tamarense (Wang et al., 2007). A sulfotransferase that specifically transfers a sulfonyl residue to a carbamoyl group present in some saxitoxin analogues has been purified and characterised from PSP toxin-producing G. catenatum (Sako et al., 2001).
The authors suggested that STX might be the only toxin directly synthesized, and that all other PSP toxins are derived by a fixed set of interconversions. Consistent with this idea, an O-sulfotransferase specifically transferring a sulfonyl group to O-22 of 11- hydroxy-STX to produce GTX 3 has also been purified and characterized from G. catenatum (Yoshida et al., 2002). If these interconversions are the same in all PSP- producing dinoflagellates, A. minutum should contain the characterized O- sulfotransferase (Yoshida et al., 2002) and the oxidase transforming GTX 3 to GTX 4
(Oshima, 1995). Searching our A minutum EST library for corresponding candidate genes revealed several oxidases and one sulfotransferase of unknown specificity. The microarray data set shows two of the potential oxidases as higher expressed in the toxic strains - one hypothetical protein similar to iron/ascorbate family oxidoreductase was between 6.6 and 10.3 times higher expressed in the toxic strains AL3T and AL9T, and a putative steroid oxidoreductase superfamily member 2.9 to 4.5 times. Both oxidases had a very good BLAST match with the PSP toxin-producers A. tamarense and A. catenella, Manuscript 1 56 respectively, but only weak or no similarities to other dinoflagellate sequences. The sulfotransferase had a potentially homologous match (BLAST e-value = 6 x 10-11) in the
A. tamarense EST library, but in none of the other dinoflagellate libraries. The microarray data did not identify it as differentially expressed between toxic and non- toxic strains. As sulfotransferases and oxidases are rather general enzyme types occurring in many other pathways including those of primary metabolism, it is not possible to determine at this point whether one of the enzymes found in A. minutum is related to PSP toxin modification.
The only available sequence information related to PSP toxin synthesis is a cyanobacterial gene cluster first identified in C. raciborskii (Kellmann et al., 2008a).
The standing hypothesis is that PSP toxin biosynthesis should follow the same pathways
(Shimizu et al., 1984) and be encoded by the same set of genes in dinoflagellates and cyanobacteria. This is usually explained by horizontal gene transfer (HGT) between the prokaryotic cyanobacteria and dinoflagellates (Shimizu, 1996; for other examples of inter-kingdom gene transfer see Lander et al., 2001; Richards et al., 2006). Although the gene cluster coding for the enzymes of the PSP synthesis pathway are known, corresponding genes in dinoflagellates have not been identified to date.
Searching our A. minutum EST library for contigs homologous to the cyanobacterial
PSP toxin genes identified several sequences of moderate similarity. However, when we calculated phylogenies from these sequence pairs and their most similar matches as identified by BLAST against SwissProt or, in the case of Amin_93i12r and
Amin_73a05f against SwissProt and nr, the A. minutum sequence and the C. raciborskii toxin gene appeared to be very distant relatives separated by many more closely related sequences and often by long branches in the Phyml-based likelihood trees. This indicates that if a suite of genes similar to the C. raciborskii PSP toxin gene cluster is Manuscript 1 57 present in the A. minutum transcriptome, none of its sequences were recovered during
EST library preparation.
A gradient PCR test using DNA from A. minutum AL3T cultures grown under antibiotic treatment and primers designed against the PSP toxin cluster from cyanobacteria recovered no PCR products except the positive control (I. Yang, unpublished data).
However, negative results in a PCR experiment cannot prove non-existence of a sequence, and other primers or different PCR conditions might lead to a different outcome. In any case, no evidence of the cyanobacterial PSP toxin gene cluster in dinoflagellates has been published, in spite of the fact that the PSP-producing
Alexandrium species are among the best-studied dinoflagellates (e.g. Hackett et al.,
2005; Jaeckisch et al., 2008; Uribe et al., 2008). Together with the data presented here, this suggests the hypothesis that the PSP toxin genes in dinoflagellates are rather more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. Whether or not this HGT event has occurred as hypothesized, or if the capacity for PSP toxin production arose independently in dinoflagellates, or if the bacterial genes were horizontally transferred to dinoflagellates but then changed during evolutionarily relevant time-spans to a point beyond recognition with current methods, can only be determined when the first dinoflagellate PSP toxin genes are identified.
Physiology-based approach
Although it has been suggested (Monroe & Van Dolah, 2008) that many genes in dinoflagellates are post-transcriptionally regulated, examination of mRNA pools regularly succeed in finding genes differentially expressed between different physiological treatments or at different times of the light-dark cycle (Taroncher-
Oldenburg & Anderson, 2000; Okamoto & Hastings, 2003b; Okamoto & Hastings,
2003a; Van Dolah et al., 2007). The ability to produce PSP toxins is genetically fixed Manuscript 1 58 within strains, and as it is probably genetically determined, evidence should be traceable to the mRNA pool, whether transcriptionally or post-transcriptionally regulated. This interpretation serves to justify our attempt to identify possible candidate genes for involvement in PSP toxin production by comparing mRNA pools of toxic and non-toxic strains.
Certain experimental efforts to understand PSP toxin production in Alexandrium species have focussed on identifying critical time-points during the cell cycle. For example, in light-deprivation synchronized cultures of Alexandrium fundyense, PSP toxin production was shown to be coupled to an 8-10 h period in the G1 phase of the cell cycle (Taroncher-Oldenburg et al., 1997). Several dinoflagellate species are reported to exhibit naturally phased cell cycles in both non-synchronized cultures and in field populations (van Dolah et al., 1995; Leighfield & Van Dolah, 2001; Van Dolah et al.,
2008). As the toxic A. minutum strain AL3T is capable of a growth rate of one cell division per day under optimal conditions, we suspected that even experimentally non- manipulated cultures might exhibit a circadian-phased (perhaps even synchronised) cell cycle similar to that found for the dinoflagellate Amphidinium operculatum (Leighfield et al., 2002). However, both cell numbers and intracellular toxin per culture volume increased continuously throughout the light phase (see Fig. 2) in non-synchronised
AL3T cultures. A similarly broad time frame for cell division in a dinoflagellate is only known for a slow-growing culture of Gyrodinium uncatenatum (Cetta & Anderson,
1990) The apparent lack of phased cell division in non-synchronised A. minutum AL3T cultures might be an artefact of growth under conditions optimised for high growth rate and therefore may not reflect growth under natural conditions or in experimentally synchronised cultures. However, the failure of most gene expression – based HCL support trees to resolve the difference between mid-day and afternoon triplicates of the
AL3T – AL1T and AL9T – AL1T- comparisons indicate that a similar lack of phased Manuscript 1 59 physiological conditions might occur in other A. minutum strains grown under the same conditions, although we did not examine the timing of cell division and toxin production in the other strains in this study. Both laboratory cultures and field population of different Alexandrium species are known to be primarily in G1 throughout most of the light period or daylight time (Taroncher-Oldenburg et al., 1997; Garcés et al., 1998; Figueroa et al., 2007). Thus, combining data from two different time-points during light phase should capture differences in the mRNA pool of toxic and non-toxic strains during PSP toxin production while excluding strain-specific differences in circadian responses not related to toxin production.
The microarray approach can identify differences in mRNA abundances of known sequences irrespective of the assigned function, to compare the gene expression of the different strains. This allows screening for gene expression patterns associated with different genetic traits coupled to physiological responses such as toxin production, and can be used to correlate non-annotatable sequences with biological connotation (Pir et al., 2006). As the two-colour microarray setup used in this study directly measures the ratio of sequence abundances in the two samples hybridized, the detection of these differences is largely independent of overall expression levels.
In inter-strain comparisons using microarrays, mRNA differences between the strains might result in differences in hybridisation efficiency which from the array data alone cannot be distinguished from expression level differences. However, comparison of microarray results (see Additional file 2:_List of differentially expressed genes) with qPCR data (Fig. 4) indicated that in this respect, our microarray setup is more likely to pick up slightly divergent sequences than the gene expression “gold standard” qPCR.
According to microarray data we identified 145 genes as higher expressed in both toxic strains at both time-points examined. Among the eight of these that were not Manuscript 1 60 significantly expressed in the non-toxic strain were two putative helicase sequences and six hypothetical proteins for which no annotation could be found. We consider these eight genes as candidates for genes associated with the biosynthesis or regulation of
PSP toxins, or for adaptive responses to intracellular PSP toxins. For one of the non- annotatable candidates we obtained a significant BLAST hit (E-value 6x10-14) against
ESTs of the PSP toxin-producing species A. tamarense, but not against any of the non-
PSP-toxin-producing dinoflagellates tested. As we do not know the proportion of the respective transcriptomes represented in either EST library, we rate the match but not the lack of matches as significant. Therefore we consider these eight genes candidates for genes associated with the biosynthesis or regulation of PSP toxicity, or for acclimatisation to intracellular PSP toxins.
The putative functions of 31 of the genes higher expressed in the toxic strains, and of 8 of the 47 genes higher expressed in the non-toxic strain AL1T, were identified by manual annotation. A number of these were involved in post-transcriptional processes, which in dinoflagellates seems to be an important level of gene regulation (Bachvaroff
& Place, 2008).
Most of the gene expression differences mentioned herein are probably due to variations in growth rate, overall metabolic rate, or other physiological differences between individual strains. Nevertheless, the genes that were not significantly expressed in the non-toxic AL1T match the expression pattern expected for genes involved in toxin biosynthesis, regulation or sequestration. We therefore propose a particular focus on these sequences as gene targets to be further comparatively studied in other dinoflagellates and species complexes for which toxigenic and non-toxigenic strains are available. All authors read and approved the final manuscript. Manuscript 1 61
Conclusions
An in-silico approach to identify genes related to the recently published cyanobacterial
PSP toxin gene cluster in our new EST library of the PSP toxin-producing dinoflagellate
A. minutum did not yield evidence for cyanobacteria-like sxt genes in our species. A semi-extensive PCR approach involving A. minutum DNA and primers designed for the cyanobacterial genes also failed to detect congruent sequences related to PSP toxin biosynthesis. Although EST libraries such as the one presented here represent only a subsample of the transcriptome, the lack of evidence for all of these genes in our library, as well as in other published EST libraries of PSP toxin-producing Alexandrium species, suggests that the PSP toxin genes in dinoflagellates are more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer.
This does not rule out a cyanobacterial ancestry for the dinoflagellate PSP toxin genes, because the possibility of rapid change of newly obtained genes in the dinoflagellate nucleus cannot be excluded.
Microarray-based comparisons of toxic and non-toxic strains of A. minutum indicated that many genes were higher expressed in the toxic than in the non-toxic strain. Among contigs candidates for PSP toxin-related genes are the several genes that could not be annotated due to a lack of known similar sequences but were expressed only in the toxic strains. Further testing of these candidates, by expression analysis with different toxic and non-toxic strains and by physiological manipulations that affect the biosynthetic rate and cell content of particular toxin analogues is clearly warranted.
While transformation of dinoflagellates has rarely been achieved (ten Lohuis & Miller,
1998), several studies have demonstrated successful expression of dinoflagellate genes in E. coli (Bae & Hastings, 1994; Suzuki-Ogoh et al., 2008) or yeast (Lippmeier et al.,
2002; Ho et al., 2007).To obtain conclusive evidence for the function of PSP toxin Manuscript 1 62 genes it might be necessary to use heterologous expression of the identified candidates in a suitable expression system, followed by purification of the enzymes and assessment of their activity on known PSP toxin precursors.
Authors’ contributions
UJ and AC conceived of the study, participated in its design and helped to draft the manuscript; UJ also participated in the day-to-day management of this study and in the design of the microarray. GG sequenced the cDNA library, constructed the EST contigs and provided part of the bioinformatic analysis. BK conceived of the LC-MS/MS measurement protocol, carried out these measurements and provided the LC-FD protocols. AG provided the SAMS part of the automated annotation. IY and SB determined the growth conditions, grew and harvested the input cultures for the EST library. SB conceived of the RNA extraction protocol. IY carried out the remainder of the molecular genetics studies, participated in the bioinformatics analysis, carried out the manual annotation and drafted the manuscript. All authors read and approved the final paper.
Acknowledgements
Thanks to Bánk Beszteri for help with the PhyloGena computations and for general bioinformatics support, to Nina Jaeckisch for access to the unpublished A. ostenfeldii
EST library, to Karina Stucken for sharing her sxt gene primers, and to Annegret Müller for running the LC-FD.
This study was financed by the EU-project ESTTAL (GOCE-CT2004-511154) and
Marine Genomics Europe (GOCE-CT-2004-505403). Manuscript 1 63
References
ALTSCHUL, S. F., MADDEN, T. L., SCHÄFFER, A. A., ZHANG, J., ZHANG, Z., MILLER, W., LIPMAN, D. J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research 25(17): 3389-3402.
ALVERCA, E., FRANCA, S., MORENO DÍAZ DE LA ESPINA, S. (2006). Topology of splicing and snRNP biogenesis in dinoflagellate nuclei. Biology Of The Cell 98(12): 709-720.
BACHVAROFF, T., PLACE, A. (2008). From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae. PLoS ONE 3(8).
BAE, Y. M., HASTINGS, J. W. (1994). Cloning, sequencing and expression of dinoflagellate luciferase DNA from a marine alga, Gonyaulax polyedra. Biochimica Et Biophysica Acta-Gene Structure and Expression 1219(2): 449-456.
CEMBELLA, A. D. (1998). Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. In Physiological Ecology of Harmful Algal Blooms (D. M. ANDERSON, A. D. CEMBELLA, G. M. HALLEGRAEFF editors), pp. 381-403. Springer Verlag, Berlin, Heidelberg, New York.
CEMBELLA, A. D., JOHN, U. (2006). Molecular physiology of toxin production and growth regulation in harmful algae. In Ecology of Harmful Algae (E. GRANÉLI , J. T. TURNER editors), pp. 215-227. Springer Berlin Heidelberg.
CETTA, C. M., ANDERSON, D. M. (1990). Cell-cycle studies of the dinoflagellates Gonyaulax polyedra Stein and Gyrodinium uncatenum Hulburt during asexual and sexual reproduction. Journal of Experimental Marine Biology and Ecology 135(1): 69-84.
CHO, Y., HIRAMATSU, K., OGAWA, M., OMURA, T., ISHIMARU, T., OSHIMA, Y. (2008). Non- toxic and toxic subclones obtained from a toxic clonal culture of Alexandrium tamarense (Dinophyceae): Toxicity and molecular biological feature. Harmful Algae 7(6): 740-751.
DIENER, M., ERLER, K., CHRISTIAN, B., LUCKAS, B. (2007). Application of a new zwitterionic hydrophillic interaction chromatography column for determination of paralytic shellfish poisoning toxins. Journal of Separation Science 30(12): 1821-1826.
ESCHBACH, E., JOHN, U., RECKERMANN, M., CEMBELLA, A. D., EDVARDSEN, B., MEDLIN, L. K. (2005). Cell cycle dependent expression of toxicity by the ichthyotoxic prymnesiophyte Chrysochromulina polylepis. Aquatic Microbial Ecology 39: 85–95.
FAST, N. M., KISSINGER, J. C., ROOS, D. S., KEELING, P. J. (2001). Nuclear-encoded, plastid- targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids. Molecular Biology And Evolution 18(3): 418-426.
FIGUEROA, R. I., GARCÉS, E., BRAVO, I. (2007). Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales, Dinophyceae) in culture. Journal of Phycology 43(5): 1039-1053.
GARCÉS, E., DELGADO, M., MASÓ, M., CAMP, J. (1998). Life history and in situ growth rates of Alexandrium taylori (Dinophyceae, Pyrrophyta). Journal of Phycology 34(5): 880-887.
GARCÍA, C., DEL CARMEN BRAVO, M., LAGOS, M., LAGOS, N. (2004). Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords. Toxicon 43(2): 149.
GERACI, J. R., ANDERSON, D. M., TIMPERI, R. J., STAUBIN, D. J., EARLY, G. A., PRESCOTT, J. H., MAYO, C. A. (1989). Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Canadian Journal Of Fisheries And Aquatic Sciences 46(11): 1895-1898. Manuscript 1 64
GUILLARD, R. R. L., RYTHER, J. H. (1962). Studies on marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervaceae (Cleve) Gran. Canadian Journal of Microbiology 8: 229-239.
GUILLARD, R. R. L. (1975). Culture of marine phytoplankton for feeding marine invertebrates. In Culture of Marine Invertebrate Animals (W. L. SMITH, M. H. CHANEY editors), pp. 29-60. Plenum, New York.
GUINDON, S., GASCUEL, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52(5): 696-704.
GUISANDE, C., FRANGÓPULOS, M., MANEIRO, I., VERGARA, A. R., RIVEIRO, I. (2002). Ecological advantages of toxin production by the dinoflagellate Alexandrium minutum under phosphorus limitation. Marine Ecology Progress Series 225: 169-176.
HACKETT, J., SCHEETZ, T., YOON, H., SOARES, M., BONALDO, M., CASAVANT, T., BHATTACHARYA, D. (2005). Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6(1): 80.
HANEKAMP, K., BOHNEBECK, U., BESZTERI, B., VALENTIN, K. (2007). PhyloGena - a user- friendly system for automated phylogenetic annotation of unknown sequences. Bioinformatics 23(7): 793- 801.
HO, P., KONG, K., CHAN, Y., TSANG, J., WONG, J. (2007). An unusual S-adenosylmethionine synthetase gene from dinoflagellates is methylated. BMC Molecular Biology 8(1): 87.
HOLD, G. L., SMITH, E. A., BIRKBECK, T. H., GALLACHER, S. (2001). Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria. FEMS Microbiology Ecology 36(2-3): 223.
ISHIDA, Y., UCHIDA, A., SAKO, Y. (1998). Genetic and biochemical approaches to PSP toxin production of toxic dinoflagellates. In Physiological Ecology of Harmful Algal Blooms (D. M. ANDERSON, A. D. CEMBELLA, G. M. HALLEGRAEFF editors), pp. 49-58. Springer-Verlag, Heidelberg.
JAECKISCH, N., GLÖCKNER, G., VOGEL, H., CEMBELLA, A., JOHN, U. (2008). Genomic characterization of the spirolide-producing dinoflagellate Alexandrium ostenfeldii with special emphasis on PKS genes. In Proceedings of 12th International Conference on Harmful Algae, Copenhagen, Denmark. 04.-08.09.2006 (Ø. MOESTRUP, G. DOUCETTE, H. ENEVOLDSEN editors)
JASTI, S., SIERACKI, M. E., POULTON, N. J., GIEWAT, M. W., ROONEY-VARGA, J. N. (2005). Phylogenetic diversity and specificity of bacteria closely associated with Alexandrium spp. and other phytoplankton. Applied and Environmental Microbiology 71(7): 3483-3494.
JOHN, U., QUILLIAM, M. A., MEDLIN, L., CEMBELLA, A. D. (2000). Spirolide production and photoperiod-dependent growth of the marine dinoflagellate Alexandrium ostenfeldii. In Proceedings of Harmful Algal Blooms 2000: proceedings of the Ninth International Conference on Harmful Algal Blooms, Hobart, Australia. 7-11 February (G. M. HALLEGRAEFF, S. I. BLACKBURN, C. J. BOLCH, R. J. LEWIS editors)
JOHN, U., MOCK, T., VALENTIN, K., CEMBELLA, A. D., MEDLIN, L. K. (2004). Dinoflagellates come from outer space but haptophytes and diatoms do not. In Proceedings of Harmful Algae 2002, St. Pete Beach, Florida, USA. 21–25.10.2002 (K. A. STEIDINGER, J. H. LANDSBERG, C. R. TOMAS, G. A. VARGO editors)
JOHN, U., BESZTERI, B., DERELLE, E., VAN DE PEER, Y., READ, B., MOREAU, H., CEMBELLA, A. (2008). Novel insights into evolution of protistan polyketide synthases through phylogenomic analysis. Protist 159(1): 21.
KELLER, M. D., SELVIN, R. C., CLAUS, W., GUILLARD, R. R. L. (1987). Media for the culture of oceanic ultraphytoplankton. Journal of Phycology 23(4): 633-638. Manuscript 1 65
KELLMANN, R., MICHALI, T. K., NEILAN, B. A. (2008a). Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers. Journal of Molecular Evolution 67(5): 526-538.
KELLMANN, R., MIHALI, T. K., JEON, Y. J., PICKFORD, R., POMATI, F., NEILAN, B. A. (2008b). Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Applied and Environmental Microbiology 74(13): 4044-4053.
KNUDSEN, B., KNUDSEN, T., FLENSBORG, M., SANDMANN, H., HELTZEN, M., ANDERSEN, A., DICKENSON, M., BARDRAM, J., STEFFENSEN, P. J., MØNSTED, S., LAURITZEN, T., FORSBERG, R., THANBICHLER, A., BENDTSEN, J. D., GÖRLITZ, L., RASMUSSEN, J., TORDRUP, D., VÆRUM, M., RAVN, M. N., HACHENBERG, C., FISKER, E., DEKKER, P., SCHULTZ, J.: CLC Sequence Viewer. C. B. A/S, version 6.0.2.
KRELL, A., FUNCK, D., PLETTNER, I., JOHN, U., DIECKMANN, G. (2007). Regulation of proline metabolism under salt stress in the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae). Journal of Phycology 43(4): 753-762.
KROCK, B., SEGUEL, C. G., CEMBELLA, A. D. (2007). Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry. Harmful Algae 6(5): 734-744.
LAJEUNESSE, T. C., LAMBERT, G., ANDERSEN, R. A., COFFROTH, M. A., GALBRAITH, D. W. (2005). Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. Journal of Phycology 41(4): 880-886.
LANDER, E. S., LINTON, L. M., BIRREN, B., NUSBAUM, C., ZODY, M. C., BALDWIN, J., DEVON, K., DEWAR, K., DOYLE, M., FITZHUGH, W., FUNKE, R., GAGE, D., HARRIS, K., HEAFORD, A., HOWLAND, J., KANN, L., LEHOCZKY, J., LEVINE, R., MCEWAN, P., MCKERNAN, K., MELDRIM, J., MESIROV, J. P., MIRANDA, C., MORRIS, W., NAYLOR, J., RAYMOND, C., ROSETTI, M., SANTOS, R., SHERIDAN, A., SOUGNEZ, C., STANGE-THOMANN, N., STOJANOVIC, N., SUBRAMANIAN, A., WYMAN, D., ROGERS, J., SULSTON, J., AINSCOUGH, R., BECK, S., BENTLEY, D., BURTON, J., CLEE, C., CARTER, N., COULSON, A., DEADMAN, R., DELOUKAS, P., DUNHAM, A., DUNHAM, I., DURBIN, R., FRENCH, L., GRAFHAM, D., GREGORY, S., HUBBARD, T., HUMPHRAY, S., HUNT, A., JONES, M., LLOYD, C., MCMURRAY, A., MATTHEWS, L., MERCER, S., MILNE, S., MULLIKIN, J. C., MUNGALL, A., PLUMB, R., ROSS, M., SHOWNKEEN, R., SIMS, S., WATERSTON, R. H., WILSON, R. K., HILLIER, L. W., MCPHERSON, J. D., MARRA, M. A., MARDIS, E. R., FULTON, L. A., CHINWALLA, A. T., PEPIN, K. H., GISH, W. R., CHISSOE, S. L., WENDL, M. C., DELEHAUNTY, K. D., MINER, T. L., DELEHAUNTY, A., KRAMER, J. B., COOK, L. L., FULTON, R. S., JOHNSON, D. L., MINX, P. J., CLIFTON, S. W., HAWKINS, T., BRANSCOMB, E., PREDKI, P., RICHARDSON, P., WENNING, S., SLEZAK, T., DOGGETT, N., CHENG, J. F., OLSEN, A., LUCAS, S., ELKIN, C., UBERBACHER, E., FRAZIER, M., GIBBS, R. A., MUZNY, D. M., SCHERER, S. E., BOUCK, J. B., SODERGREN, E. J., WORLEY, K. C., RIVES, C. M., GORRELL, J. H., METZKER, M. L., NAYLOR, S. L., KUCHERLAPATI, R. S., NELSON, D. L., WEINSTOCK, G. M., SAKAKI, Y., FUJIYAMA, A., HATTORI, M., YADA, T., TOYODA, A., ITOH, T., KAWAGOE, C., WATANABE, H., TOTOKI, Y., TAYLOR, T., WEISSENBACH, J., HEILIG, R., SAURIN, W., ARTIGUENAVE, F., BROTTIER, P., BRULS, T., PELLETIER, E., ROBERT, C., WINCKER, P., ROSENTHAL, A., PLATZER, M., NYAKATURA, G., TAUDIEN, S., RUMP, A., YANG, H. M., YU, J., WANG, J., HUANG, G. Y., GU, J., HOOD, L., ROWEN, L., MADAN, A., QIN, S. Z., DAVIS, R. W., FEDERSPIEL, N. A., ABOLA, A. P., PROCTOR, M. J., MYERS, R. M., SCHMUTZ, J., DICKSON, M., GRIMWOOD, J., COX, D. R., OLSON, M. V., KAUL, R., RAYMOND, C., SHIMIZU, N., KAWASAKI, K., MINOSHIMA, S., EVANS, G. A., ATHANASIOU, M., SCHULTZ, R., ROE, B. A., CHEN, F., PAN, H. Q., RAMSER, J., LEHRACH, H., REINHARDT, R., MCCOMBIE, W. R., DE LA BASTIDE, M., DEDHIA, N., BLÖCKER, H., HORNISCHER, K., NORDSIEK, G., AGARWALA, R., ARAVIND, L., BAILEY, J. A., BATEMAN, A., BATZOGLOU, S., BIRNEY, E., BORK, P., BROWN, D. G., BURGE, C. B., CERUTTI, L., CHEN, H. C., CHURCH, D., CLAMP, M., COPLEY, R. R., DOERKS, T., EDDY, S. R., EICHLER, E. E., FUREY, T. S., GALAGAN, J., GILBERT, J. G. R., HARMON, C., HAYASHIZAKI, Y., HAUSSLER, D., HERMJAKOB, H., HOKAMP, K., JANG, W. H., JOHNSON, L. S., JONES, T. A., KASIF, S., KASPRYZK, A., KENNEDY, S., KENT, W. J., KITTS, P., KOONIN, E. V., KORF, I., KULP, D., LANCET, D., LOWE, T. M., MCLYSAGHT, A., MIKKELSEN, T., MORAN, J. V., MULDER, N., POLLARA, V. J., PONTING, C. P., SCHULER, G., SCHULTZ, J. R., SLATER, G., Manuscript 1 66
SMIT, A. F. A., STUPKA, E., SZUSTAKOWKI, J., THIERRY-MIEG, D., THIERRY-MIEG, J., WAGNER, L., WALLIS, J., WHEELER, R., WILLIAMS, A., WOLF, Y. I., WOLFE, K. H., YANG, S. P., YEH, R. F., COLLINS, F., GUYER, M. S., PETERSON, J., FELSENFELD, A., WETTERSTRAND, K. A., PATRINOS, A., MORGAN, M. J. (2001). Initial sequencing and analysis of the human genome. Nature 409(6822): 860-921.
LE, S. Q., GASCUEL, O. (2008). An improved general amino acid replacement matrix. Molecular Biology And Evolution 25(7): 1307-1320.
LEIGHFIELD, T. A., VAN DOLAH, F. M. (2001). Cell cycle regulation in a dinoflagellate, Amphidinium operculatum: identification of the diel entraining cue and a possible role for cyclic AMP. Journal of Experimental Marine Biology and Ecology 262(2): 177-197.
LEIGHFIELD, T. A., BARBIER, M., VAN DOLAH, F. M. (2002). Evidence for cAMP-dependent protein kinase in the dinoflagellate, Amphidinium operculatum. Comparative Biochemistry And Physiology B-Biochemistry & Molecular Biology 133(3): 317-324.
LEVY, M. G., LITAKER, R. W., GOLDSTEIN, R. J., DYKSTRA, M. J., VANDERSEA, M. W., NOGA, E. J. (2007). Piscinoodinium, a fish-ectoparasitic dinoflagellate, is a member of the class Dinophyceae, subclass Gymnodiniphycidae: Convergent evolution with Amyloodinium. Journal Of Parasitology 93(5): 1006-1015.
LIDIE, K. B., RYAN, J. C., BARBIER, M., VAN DOLAH, F. M. (2005). Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of DNA microarray. Marine Biotechnology 7(5): 481-493.
LIDIE, K. B., VAN DOLAH, F. M. (2007). Spliced leader RNA-mediated trans-splicing in a dinoflagellate, Karenia brevis. Journal Of Eukaryotic Microbiology 54(5): 427-435.
LILLY, E. L., HALANYCH, K. M., ANDERSON, D. M. (2005). Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group. Harmful Algae 4(6): 1004–1020.
LIPPMEIER, J. C., BROWN, A. M., APT, K. E. (2002). Isolation of algal genes by functional complementation of yeast. Journal of Phycology 38(3): 529-533.
LU, Y. H., CHAI, T. J., HWANG, D. F. (2000). Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity. Journal of Natural Toxins 9(4): 409-417.
MCEWAN, M., HUMAYUN, R., SLAMOVITS, C. H., KEELING, P. J. (2008). Nuclear genome sequence survey of the dinoflagellate Heterocapsa triquetra. Journal Of Eukaryotic Microbiology 55(6): 530-535.
MIHALI, T., KELLMANN, R., NEILAN, B. (2009). Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. BMC Biochemistry 10(1): 8.
MIN, X. J., BUTLER, G., STORMS, R., TSANG, A. (2005). OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Research 33: W677-W680.
MONROE, E. A., VAN DOLAH, F. M. (2008). The toxic dinoflagellate Karenia brevis encodes novel type I-like polyketide synthases containing discrete catalytic domains. Protist 159(3): 471-482.
MONTRESOR, M., JOHN, U., BERAN, A., MEDLIN, L. K. (2004). Alexandrium tamutum sp nov (Dinophyceae): A new nontoxic species in the genus Alexandrium Journal of Phycology 40(2): 398-411.
MORENO DÍAZ DE LA ESPINA, S., ALVERCA, E., CUADRADO, A., FRANCA, S. (2005). Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates. European Journal of Cell Biology 84(2-3): 137-149.
OKAMOTO, O. K., HASTINGS, J. W. (2003a). Novel dinoflagellate clock-related genes identified through microarray analysis. Journal of Phycology 39(3): 519-526. Manuscript 1 67
OKAMOTO, O. K., HASTINGS, J. W. (2003b). Genome-wide analysis of redox-regulated genes in a dinoflagellate. Gene 321: 73.
OSHIMA, Y. (1995). Chemical and enzymatic transformation of paralytic shellfish toxins in marine organisms. In Harmful Marine Algal Blooms (P. LASSUS, G. ARZUL, E. ERARD, P. GENTIEN, C. MARCAILLOU editors), pp. 475-480. Lavoisier, Paris (France).
PATRON, N. J., WALLER, R. F., ARCHIBALD, J. M., KEELING, P. J. (2005). Complex protein targeting to dinoflagellate plastids. Journal of Molecular Biology 348(4): 1015.
PIR, P., ULGEN, K. O., HAYES, A., ONSAN, Z. I., KIRDAR, B., OLIVER, S. G. (2006). Annotation of unknown yeast ORFs by correlation analysis of microarray data and extensive literature searches. Yeast 23(7): 553-571.
RICHARDS, T. A., DACKS, J. B., JENKINSON, J. M., THORNTON, C. R., TALBOT, N. J. (2006). Evolution of filamentous plant pathogens: Gene exchange across eukaryotic kingdoms. Current Biology 16(18): 1857-1864.
RUDD, S. (2003). Expressed sequence tags: alternative or complement to whole genome sequences? Trends In Plant Science 8(7): 321-329.
SAEED, A. I., BHAGABATI, N. K., BRAISTED, J. C., LIANG, W., SHAROV, V., HOWE, E. A., LI, J., THIAGARAJAN, M., WHITE, J. A., QUACKENBUSH, J. (2006). TM4 microarray software suite. Methods in Enzymology Volume 411: 134-193.
SAKO, Y., YOSHIDA, T., UCHIDA, A., ARAKAWA, O., NOGUCHI, T., ISHIDA, Y. (2001). Purification and characterization of a sulfotransferase specific to N-21 of saxitoxin and gonyautoxin 2+3 from the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae). Journal of Phycology 37(6): 1044- 1051.
SALDARRIAGA, J. F., TAYLOR, F. J. R., KEELING, P. J., CAVALIER-SMITH, T. (2001). Dinoflagellate nuclear SSU rRNA phylogeny suggests multiple plastid losses and replacements. Journal of Molecular Evolution 53(3): 204.
SCHOLIN, C. A., HALLEGRAEFF, G. M., ANDERSON, D. M. (1995). Molecular evolution of the Alexandrium tamarense species complex (Dinophyceae)-dispersal in the North American and West Pacific regions. Phycologia 34(6): 472-485.
SELANDER, E., THOR, P., TOTH, G., PAVIA, H. (2006). Copepods induce paralytic shellfish toxin production in marine dinoflagellates. Proceedings Of The Royal Society B-Biological Sciences 273(1594): 1673-1680.
SHIMIZU, Y., NORTE, M., HORI, A., GENENAH, A., KOBAYASHI, M. (1984). Biosynthesis of saxitoxin analogs: the unexpected pathway. Journal of the American Chemical Society 106(21): 6433- 6434.
SHIMIZU, Y. (1996). Microalgal metabolites: a new perspective. Annual Review of Microbiology 50: 431-465.
STOREY, J. D. (2003). The positive false discovery rate: A Bayesian interpretation and the q-value. Annals Of Statistics 31(6): 2013-2035.
STUCKEN, K., JOHN, U., CEMBELLA, A., MURILLO, A. A., SOTO-LIEBE, K., FUENTES- VALDÉS, J. J., FRIEDEL, M., PLOMINSKY, A. M., VÁSQUEZ, M., GLÖCKNER, G. (2010). The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications. PLoS ONE 5(2): e9235.
SUZUKI-OGOH, C., WU, C., OHMIYA, Y. (2008). C-terminal region of the active domain enhances enzymatic activity in dinoflagellate luciferase. Photochemical & Photobiological Sciences 7(2): 208-211. Manuscript 1 68
TANIKAWA, N., AKIMOTO, H., OGOH, K., CHUN, W., OHMIYA, Y. (2004). Expressed sequence tag analysis of the dinoflagellate Lingulodinium polyedrum during dark phase. Photochemistry and Photobiology 80(1): 31-35.
TARONCHER-OLDENBURG, G., KULIS, D. M., ANDERSON, D. M. (1997). Toxin variability during the cell cycle of the dinoflagellate Alexandrium fundyense. Limnology and Oceanography 42: 1178-1188.
TARONCHER-OLDENBURG, G., ANDERSON, D. M. (2000). Identification and characterization of three differentially expressed genes, encoding S-adenosylhomocysteine hydrolase, methionine aminopeptidase, and a histone-like protein, in the toxic dinoflagellate Alexandrium fundyense. Applied and Environmental Microbiology 66(5): 2105-2112.
TEN LOHUIS, M. R., MILLER, D. J. (1998). Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. The Plant Journal 13: 427.
TILLMANN, U., JOHN, U. (2002). Toxic effects of Alexandrium spp. on heterotrophic dinoflagellates: an allelochemical defence mechanism independent of PSP-toxin content. Marine Ecology-Progress Series 230: 47-58.
TILLMANN, U., ALPERMANN, T., JOHN, U., CEMBELLA, A. (2008). Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists. Harmful Algae 7(1): 52.
TOUZET, N., FRANCO, J. M., RAINE, R. (2007). Characterization of nontoxic and toxin-producing strains of Alexandrium minutum (Dinophyceae) in Irish coastal waters. Applied and Environmental Microbiology 73(10): 3333-3342.
TRAINER, V. L., BADEN, D. G. (1999). High affinity binding of red tide neurotoxins to marine mammal brain. Aquatic Toxicology 46(2): 139-148.
TURNER, J. T., TESTER, P. A. (1997). Toxic marine phytoplankton, zooplankton grazers, and pelagic food webs. Limnology and Oceanography 42(5): 1203-1214.
TUSHER, V. G., TIBSHIRANI, R., CHU, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proceedings of the National Academy of Sciences of the United States of America 98(9): 5116-5121.
URIBE, P., ESPEJO, R. T. (2003). Effect of associated bacteria on the growth and toxicity of Alexandrium catenella. Applied and Environmental Microbiology 69(1): 659-662.
URIBE, P., FUENTES, D., VALDES, J., SHMARYAHU, A., ZUNIGA, A., HOLMES, D., VALENZUELA, P. D. T. (2008). Preparation and analysis of an expressed sequence tag library from the toxic dinoflagellate Alexandrium catenella Marine Biotechnology 10(6): 692-700.
VAN DOLAH, F. M., LEIGHFIELD, T. A., SANDEL, H. D., HSU, C. K. (1995). Cell division in the dinoflagellate Gamierdiscus toxicus is phased to the diurnal cycle and accompanied by activation of the cell cycle regulatory protein, CDC2 kinase. Journal of Phycology 31(3): 395-400.
VAN DOLAH, F. M., LIDIE, K. B., MOREY, J. S., BRUNELLE, S. A., RYAN, J. C., MONROE, E. A., HAYNES, B. L. (2007). Microarray analysis of diurnal- and circadian-regulated genes in the Florida red- tide dinoflagellate Karenia brevis (Dinophyceae). Journal of Phycology 43(4): 741-752.
VAN DOLAH, F. M., LEIGHFIELD, T. A., KAMYKOWSKI, D., KIRKPATRICK, G. J. (2008). Cell cycle behavior of laboratory and field populations of the Florida red tide dinoflagellate, Karenia brevis. Continental Shelf Research 28(1): 11-23.
WANG, D., ZHANG, S., HONG, H. (2007). A sulfotransferase specific to N-21 of gonyautoxin 2/3 from crude enzyme extraction of toxic dinoflagellate Alexandrium tamarense CI01. Chinese Journal of Oceanology and Limnology 25(2): 227. Manuscript 1 69
WYATT, T., JENKINSON, I. (1997). Notes on Alexandrium population dynamics. Journal Of Plankton Research 19(5): 551-575.
YOSHIDA, T., SAKO, Y., UCHIDA, A., KAKUTANI, T., ARAKAWA, O., NOGUCHI, T., ISHIDA, Y. (2002). Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae). Fisheries Science 68(3): 634-642.
ZHANG, H., HOU, Y. B., MIRANDA, L., CAMPBELL, D. A., STURM, N. R., GAASTERLAND, T., LIN, S. J. (2007). Spliced leader RNA trans-splicing in dinoflagellates. Proceedings of the National Academy of Sciences of the United States of America 104(11): 4618-4623. Manuscript 1 70
Figures
Figure 1 KOG-based functional classification of 1005 contigs as obtained by BLAST- identified similarities Manuscript 1 71
Figure 2 Increase of cell number and culture toxin content in strain AL3T over 24 hours. Grey boxes =dark phase, circles = cell numbers, diamonds = total PSP toxins mL-1. Manuscript 1 72
Figure 3 Extracted ion traces of LC-MS/MS chromatograms of the A. minutum strains
AL1T, AL3T and AL9T. Top: transition 396 > 288 for GTX2/3 (retention time GTX3:
14.1 min); bottom: transition 412 > 314 for GTX1/4 (retention time GTX4 13.3 min) Manuscript 1 73
Figure 4 Comparison of expression ratios based on microarray- and qPCR- data.
Array data: more than 15x higher expressed in toxic strains, ++; more than 5x, +; more than 5x higher expressed in non-toxic strain, -; 2-5x higher expressed in non-toxic strain, (-); raw data see supplementary table 1. qPCR: Ct < 25, ++; Ct<27, +; Ct>27, -;
N.D., not detected at Ct < 35. Manuscript 1 74
Tables
Table 1 Base composition of the A. minutum EST library
no. of fraction of GC content contigs contigs <40% 75 1.7% 40-50% 243 5.6% 50-60% 3454 80.0% 548 12.7% 22% minimum 66% maximum
Table 2 Result of BLAST search of A. minutum contigs against all dinoflagellate ESTs available in GenBank (as of 28.11.2007).
E-value all available A. tamarense A. catenella A. ostenfeldii cut-off dinoflagellates -5 10 2209 (51.1%) 1487 (34.4%) 940 (21.8%) 1543 (35.7%) -10 10 1864 (43.1%) 1203 (27.8%) 715 (16.6%) 1335 (30.9%) -30 10 1123 (26.0%) 703 (16.3%) 360 (8.3%) 867 (20.1%)
Table 3 Strain-specific toxin content as measured by LC-FD (in fmol cell-1 ± standard deviation, n=3)
strain Aggregate toxins GTX 1/4 GTX 2/3 STX
AL3T 1.11 ± 0.06 1.09 ± 0.06 0.02 ± 0.003 traces
AL9T 10.33 ± 1.95 10.02 ± 1.94 0.29 ± 0.01 0.02 ± 0.001
AL1T 0 0 0 0 Manuscript 1 75
Table 4 Putative functions of differentially expressed genes
Contig name Gene product Putative Log mean fold changes function AL3T / AL9T / Tox / AL1T AL1T Nontox Higher expressed in toxic strains Amin_34h03r putative small nuclear gene 1.7 2.4 2.0 ribonucleoprotein expression polypeptide E Amin_36k19f translation elongation gene 24.4 25.8 25.1 factor-like protein expression Amin_44h09f glycyl-tRNA synthetase gene 4.3 4.6 4.5 expression Amin_56k21r probable translation gene 22.6 28.5 25.4 initiation factor E4 expression Amin_65d02r putative Ubiquitin-like gene 3.1 2.3 2.7 domain-containing CTD expression phosphatase 1 Amin_75e04r putative helicase gene 24.3 26.1 25.2 expression Amin_48i15f putative helicase gene 26.9 44.8 34.7 expression Amin_56g23f putative Alpha-L- protein 1.8 5.5 3.2 fucosidase 1 glycosylation Amin_95c08r sialyltransferase protein 20.3 23.5 21.9 glycosylation Amin_87p16r casein family protein signal 9.7 13.0 11.2 kinase transduction Amin_98a08r endonuclease / intracellular 3.6 4.8 4.2 exonuclease / signalling phosphatase family protein Amin_63e03r hypothetical protein cell cycle 3.7 3.2 3.4 similar to Sentrin-specific control protease 8 Amin_68d07f rrm family protein similar cell cycle 2.9 2.5 2.7 to mei2 control Amin_63c06r RNA-binding protein cell cycle 1.9 2.6 2.2 similar to mei2 control Amin_53e04r chlorophyll a-c binding chloroplast 5.5 4.9 5.2 protein Amin_81i24r translation elongation organellar 37.4 45.2 41.1 factor P translation Amin_97a05r putative mitochondrial mitochondrial 4.6 5.8 5.1 import receptor subunit tom40 Amin_70g03r ferrochelatase mitochondrial 3.0 2.8 2.9 Amin_40e21r putative hydrolase hydrolytic 5.5 3.1 4.1 enzyme Amin_82n24r phospholipase/carboxyles hydrolytic 7.2 8.8 8.0 terase family protein enzyme Amin_46a02r Abhydrolase domain- hydrolytic 5.6 4.0 4.7 Manuscript 1 76
containing protein enzyme Amin_95b07r putative sulfatase, similar hydrolytic 3.2 4.2 3.7 to Ats family enzyme arylsulfatases Amin_79g11r hypothetical protein hydrolytic 2.1 3.4 2.7 similar to various enzyme hydrolytic enzymes Amin_62h07r hypothetical protein detoxification 4.7 7.0 5.8 similar to glutathione S- transferase Amin_12c01r aminotransferase, class I Amino- 2.1 3.0 2.5 or II transferase Amin_93i12r hypothetical protein Amino- 2.5 4.2 3.3 similar to branched- transferase chain-amino-acid aminotransferase Amin_46c04r hypothetical protein Oxido- 7.9 8.8 8.3 similar to Iron/ascorbate reductase family oxidoreductases Amin_83a03f2 galactose-1-phosphate sugar 5.2 8.6 6.7 uridylyltransferase metabolism Amin_44a04r putative steroid secondary 3.2 4.0 3.6 oxidoreductase metabolites superfamily member Amin_53f01f putative lipolytic enzyme, lipid 3.5 2.3 2.8 G-D-S-L family metabolism Amin_17d04f putative CorA-like Mg2+ metal ion 3.5 3.7 3.6 transporter protein transport Amin_59d02r GFA family protein putative 20.4 22.8 21.6 carbon-sulphur lyase
Higher expressed in non-toxic strain AL1T Amin_30a05r cyclin-dependent signal -3.9 -2.9 -3.3 kinases regulatory transduction subunit 1 Amin_74g08f dynein heavy chain intracellular -5.5 -4.3 -4.9 family protein transport Amin_44h03r fibronectin type III putatively -9.2 -11.3 -10.2 domain-containing involved in protein signalling Amin_09b02f glutaredoxin family DNA -4.7 -3.2 -3.9 protein nucleotide synthesis Amin_89h11r hypothetical protein stress response -2.4 -3.2 -2.8 containing a putative 'Cold-shock' DNA- binding domain Amin_101h04r inorganic H+ proton pump -3.0 -1.9 -2.4 pyrophosphatase, 3'- region. Amin_16h04r peptide chain release translation -8.5 -7.5 -8.0 factor 1 Manuscript 1 77
Amin_42k04f hypothetical protein putative -2.7 -29.7 -9.0 similar to taurine dioxygenase catabolism dioxygenase TauD Manuscript 1 78
Additional files
Additional file 1: Primer tables and respective PCR conditions (see appendix)
All primers used and the respective PCR protocols of the study are listed in this file.
Additional file 2: List of differentially expressed genes (see file)
Table of contigs identified as differentially expressed between both toxic strains and the non-toxic strain at two time-points during light phase. Included are contig IDs, microarray-based gene expression data, characterisation of the contigs, manual annotation and results of various database searches.
Additional file 3: Phyml-based likelihood trees with bootstrap support values (see appendix)
14 phylogenies of A. minutum EST contigs that produced significant (e<10-4) BLAST hits with cyanobacterial sxt-related genes. Phylogenies were calculated including the A. minutum contig sequence, the corresponding cyanobacterial gene, and their closest
SwissProt matches as identified by PhylogGena (Top10Select-mode). Phylogenies M.2 and N.2 additionally include the best 20 hits produced by blasting the Alexandrium sequences against the NCBI non-redundant protein sequence database (nr).
Additional file 4: Rarefaction curve (see appendix)
Generated ESTs and assembled cluster as contigs were analysed using http://www.biology.ualberta.ca/jbrzusto/rarefact.php#Calculator. Manuscript 2 79
6 Manuscript 2 Manuscript 2 80
Grazer induced toxin formation in dinoflagellates:
A transcriptomic model study
Ines Yang1,2§, Erik Selander3, Henrik Pavia4 & Uwe John1
1 Ecological Chemistry, Div. Biosciences, Alfred Wegner Institute for Polar and Marine
Research, Postfach 12 01 61, 27515 Bremerhaven, Germany
2 present address: School of Biosciences, University of Exeter, Stocker Road, Exeter,
EX4 4QD, UK
3 Department of Marine Ecology - Kristineberg, University of Gothenburg, SE 450 34
Fiskebäckskil, Sweden
4 Department of Marine Ecology - Tjärnö Unviersity of Gothenburg SE 452 96
Strömstad, Sweden
§Corresponding author
Ines Yang
[email protected] Manuscript 2 81
Abstract
The cosmopolitan marine dinoflagellate Alexandrium minutum is known to produce paralytic shellfish poisoning (PSP)-causing toxins. As has recently been shown, this toxicity can be induced to higher levels by the presence of certain copepod species.
Inducible defences are known from a variety of marine organisms, but the associated transcriptomic changes have only been investigated in model angiosperms. Here, we use a microarray approach to investigate the changes in gene expression during the copepod-provoked induction of higher toxicity in A. minutum. We found a very limited set of 14 genes (0.35% of the tested sequences) to be affected by copepod presence; gene expression changed by a factor of 1.9 to 11.4 when compared to control samples.
We compared the differentially expressed genes after one day - before the onset of augmented toxicity – and after 3 days, when cellular toxin content in the induced samples was 5 times higher than in control cultures. Comparison to previously found differences between toxic and non-toxic strains revealed two sequences apparently associated with PSP toxin content. Our study is the first investigation into the transcriptomal effects associated with grazer-induced induction of elevated toxicity in a protist. This provides a novel approach to study the ecology of predator-prey interactions, as well as a highly targeted method to investigate the genes associated with toxin production and regulation in toxic dinoflagellates. Manuscript 2 82
Introduction
The potent sodium channel blocking neurotoxins collectively known as paralytic shellfish toxins (PSTs), including the well-known saxitoxin, are produced by at least eleven different species in the marine dinoflagellate genus Alexandrium (Hansen et al.,
2003), the dinoflagellates Gymnodinium catenatum and Pyrodinium bahamense as well as by some fresh water cyanophyceans (e.g. Aphanizomenon flos-aqua).
A grazer-deterring function of dinoflagellate toxins has long been suggested, but as the specific interaction strongly depends on both the toxin composition of the prey and on the grazer tested (for a review see Turner & Tester, 1997), this effect has remained controversial (Cembella, 2003). Recent results hint to a complex interplay between dinoflagellate toxicity and grazer adaptation. Colin and Dam (2002) investigated PSP toxin susceptibility in copepods from different populations and found a detrimental effect of PSP toxin-producing, but not of non-toxic, Alexandrium strains on the ingestion and egg production rates of copepods from populations not historically exposed to toxic Alexandrium blooms. Copepods from historically exposed populations were less affected and able to acclimate to the exposure within a few days. Investigating the ingestion rate of Alexandrium cells added to natural plankton samples, Teegarden et al. (2008) showed 3 of 4 tested copepod species to preferentially ingest non-toxic
Alexandrium, but to avoid PSP-producing species. Teegarden et al. (2008) suggest the magnitude of this response is dependent on both the Alexandrium cellular toxicity and concentration relative to non-toxic prey items. Further support for a grazer-deterring function of PSP toxins was provided by Selander et al (2006) who found that PST- producing dinoflagellates increase their cellular toxin content in response to waterborne cues from zooplankton grazers. Naturally occurring densities of copepods may provoke a more than twenty-five fold increase in toxicity (Bergkvist et al., 2008) in Alexandrium Manuscript 2 83 minutum, which has also been shown to correlate with increased resistance to copepod grazing (Selander et al., 2006). Besides its ecological implications for planktonic interactions and the formation of toxic algal blooms, this induction of toxin production allows for a new type of experimental design in which genetically identical algal cultures can be induced to different levels of toxin content. In combination with the recent development in genomic resources for Alexandrium spp. (Mansucript 1) it also offers a new approach to identify elicitor-sensitive genes and in particular the genes involved in toxin formation.
The cyanobacterial PST gene cluster (sxt cluster) recently identified in
Cylindrospermopsis raciborskii (Kellmann et al., 2008b) revealed a complex phyolgenetic pattern suggesting that its assembly involved both native genes and multiple horizontal gene transfers (HGTs) from different prokaryotic sources (Moustafa et al., 2009). While several recent publications deal with the identity and evolution of the sxt genes in cyanobacteria (e.g. Kellmann et al., 2008a; Mihali et al., 2009;
Moustafa et al., 2009; Stucken et al., 2010) the known similarities to dinoflagellate genes remain extremely sparse. So far, the only published similarities in dinoflagellates refer to sxtA, a cyanobacterial putative polyketide synthase. Two different parts of this saxitoxin cluster gene were found to be somewhat similar to two dinoflagellate EST, one from an A. catenella library and one from A. tamarense (Moustafa et al., 2009). At the time of the study by Moustafa et al. (2009), several large-scale sequence databases for PST-producing Alexandrium species were available (Hackett et al., 2005; Erdner &
Anderson, 2006; Uribe et al., 2008), so the sparseness of identified similarities might be due to a considerable change in the sxt genes after their HGT to dinoflagellates. During the long evolutionary timescale the sxt genes might have changed beyond recognition.
In a recent experiment (Mansucript 1), we used an in silico approach to identify homologs of the sxt gene cluster in a normalised A. minutum cDNA library sequenced to Manuscript 2 84 approx. 80% saturation. Although we found several sequences with similarities to the different sxt genes, phylogenetic analysis demonstrated all potential homologs to be closer related to sequences of organisms not known to produce PSTs. While these data are not completely conclusive, they suggest that the dinoflagellate PSP toxin genes might be too different from their cyanobacterial counterparts to be easily recognizable, and that similarity-independent ways of identifying these genes in dinoflagellates might be more promising.
Attempts to identify these genes from dinoflagellate gene expression patterns can be based either on comparisons between strains of different toxicity, or on physiological conditions affecting PSP toxin accumulation (Plumley, 1997; Cembella & John, 2006).
The main challenge in both these approaches is the inherent difficulty in discerning the differences leading to the different toxin levels from those due to unrelated physiological effects. The predator-induced differences in PSP toxin accumulation provide a system in which samples of the same strain with substantially different toxin quotas can be compared without resorting to severe P-starvation to increase toxin cell quotas or to other harsh conditions known to lead to a reduction in PSP toxin content
(Flynn et al., 1994; Hwang & Lu, 2000; Touzet et al., 2007).
The goal of this study was to identify A. minutum genes regulated by copepod presence and associated with the copepod-stimulated induction of higher toxin cell quotas. In order to do so, we employed EST-based oligonucleotide microarrays to compare the gene expression in copepod-challenged and untreated cultures. As the first gene expression analysis of grazer-induced toxin formation in phytoplankton primary producers, this study demonstrates the capability of this method to identify elicitor responsive genes in phytoplankton-grazer interactions. Manuscript 2 85
Materials and Methods
Induction experiment
We used A. minutum strain GUMACC #83 (synonymous with CCMP 113 Al and Al
1V), which originates from the Ria de Vigo in NW Spain (Franco et al., 1994), well within the distribution range of C. typicus (Beaugrand et al., 2007). A culture of this strain was diluted with K/10 medium (Keller et al., 1987) to an initial concentration of
8800 cells ml-1 and divided between 12 flasks. Each flask received 500 mL culture and half of them were supplemented with four adult female copepods (Centropages typicus). The flasks were incubated in a temperature and light controlled room (18ºC,
16:8 h light:dark cycles, ~150 µmol m-2 s-1 fluorescent light). Half of the replicates
(three controls and three copepod containing bottles) were harvested after one day, the other half received a small volume of K medium (50 ml) on day two to avoid nutrient limitation and was harvested on day three. Day three cell counts were corrected for the additional dilution. At each sampling occasion a well mixed sample of 60 mL was extracted from each replicate and analysed with triplicate counts on a particle counter
(Elzone 180XY). Based on the particle counts, duplicate samples containing at least
2*106 cells (200 mL at the Day 1 sampling occasion and 150 mL at the Day 3 sampling occasion) were gently prefiltered through a 67 µm nylon plankton mesh to eliminate copepods and copepod eggs, before vacuum filtration onto 47 mm nucleopore filters (8
µm). Each sample was rinsed twice with approximately 50 mL autoclaved filtered seawater, the filter was vacuum dried and immediately transferred to 50 mL centrifuge tubes with forceps and plunged into liquid nitrogen. The samples were stored in a -80ºC freezer until RNA extraction. A known volume of the remaining culture was suction filtered onto 25 mm glass-fibre filters, lyophilized and stored frozen until toxin analysis. Manuscript 2 86
5 mL nutrient samples were taken from the remaining volume filtered through a 0.45
µm syringe filter and stored frozen until analysis.
Toxin and nutrient analysis
PST samples from the bioassays were extracted with 1 mL 0.05 M acetic acid (aq) through three consecutive freeze-thaw cycles. The extract was filtered (Whatman GF/A) and frozen in HPLC glass vials until analysis with high performance liquid chromatography with fluorescence detection [HPLC-FD (Asp, 2004 #104)]. HPLC analyses were carried out on a Hitachi-7000 system equipped with a Genesis C8
FROXPQ 9\PDF ȝP PP *RQ\DXWR[LQ -4) standards were from NRC-
CNRC, Halifax, Canada. The A. minutum strain used here is only known to produce
GTX 1/4 and GTX 2/3 (Franco et al., 1994; Selander et al., 2006), which can be completely separated by an isocratic elution with 50 mM ammonium-phosphate buffer
(pH 7.1) and 2 mM sodiumheptanesulfonate at 0.4 mL min-1. After the separation, toxins were oxidized with 7 mM periodic acid in 50 mM sodium phosphate buffer (pH
9.0, 0.2 mL min-1) in a PEEK capillary (10 m, 80°C). The oxidation was terminated with 0.5 M acetic acid (0.2 mL min-1) before fluorescent detection at λex = 330 nm,
λem = 390 nm.
Differences in cell specific total toxicity were analysed with a two factor ANOVA, with the fixed orthogonal factors time (two levels; one and three days) and treatment (two levels; copepod present or absent). The Student-Newman-Keul post hoc procedure was used to evaluate significant differences between groups. Toxin profiles were compared between treatments by comparing the ratio between the epimers GTX 1/4 and GTX 2/3 on Day 1 and Day 3 using a two-tailed t-test with α set to 0.025 after Bonferroni correction for multiple comparisons. Manuscript 2 87
Nutrient concentrations (nitrate plus nitrite, phosphate) were determined using standard methods (Strickland & Parsons, 1972) to verify that the cultures were not nutrient limited during experiments.
Microarray analysis
RNA extraction followed a modified TriReagent protocol (Sigma-Aldrich, Steinheim,
Germany) with additional cleaning steps. Cells were briefly lysed by 10 min incubation at 60°C in TriReagent, aided by repeated vortexing with glass beads included in the sample tube. After addition of 200 µL chloroform per mL TriReagent, samples were centrifuged for 15 min at 12000 g and 4°C. The aqueous phase was mixed with an equal volume of isopropanol and incubated at -20°C for at least 10 minutes. An RNA pellet was obtained by centrifugation at 12 000 g for 10 min at 4°C. It was washed by addition of 75% ethanol, followed by another centrifugation step. After removal of the ethanol, the pellet was allowed to dry until hyaline and then dissolved in 100 µL RNAse-free water (Qiagen, Hilden, Germany). RNA cleanup and DNA digestion followed the protocol supplied with the Qiagen RNeasy kit: RNA samples were mixed with 350 µL binding buffer RLT (Qiagen) containing 1% ȕ-mercaptoehtanol. After mixing with 250
µL ethanol, samples were applied to an RNeasy column (Qiagen) containing a silica membrane. Columns were washed by 1 min incubation with 700 µL RW1 and centrifugation, before 10 µL DNase I mixed with 70 µL Buffer RDD (both Qiagen) were applied for 15 min. To interrupt DNase digestion, columns were washed with 700
µL RW1. Samples were incubated for 1 min in Buffer RPE (Qiagen), centrifuged, and washed again with the same buffer. After 2 minutes centrifugation and another 1 min high-speed centrifugation in a new collection tube, RNA was eluded with 40 µL RNase- free water (Qiagen). To increase final RNA concentration, the flow-through was applied to the membrane a second time. Where necessary, an additional cleanup and Manuscript 2 88 concentration step using Microcon Ultracel YM-30 columns (Millipore, Schwalbach,
Germany) was applied. RNA purity and quantity were determined using a NanoDrop
(PeqLab, Erlangen, Germany), RNA integrity was assessed using a Bioanalyzer
(Agilent Technologies, Böblingen, Germany). Total RNA (250 ng/sample) was amplified and labelled using an Agilent low-input linear amplification kit. Microarrays were scanned on a Agilent G2565AA scanner, and raw data was extracted with the
Agilent Feature Extraction Software version 9.1.3.1 (FE). Array quality was monitored using the Agilent QC Tool (v1.0) with the metric set GE2_QCMT_Feb07.
Pre-processed data were subject to SAM (Significance Analysis of Microarrays (Tusher et al., 2001)) as implemented in MeV 4.0 (Saeed et al., 2003), and SAM-based q-values
(Storey, 2003) were calculated. Probes with a q-value of <1% were considered to indicate differential expression of the corresponding genes if the mean fold change of the sample triplicate was at least 1.5.
Results
Cell-specific toxin content was not significantly different after one day of incubation
(p=0.32 according to Student–Newman–Keul’s multiple comparison procedure (SNK)).
After three days however, the toxin content per cell was approximately five times higher in the copepod-exposed cultures compared to controls (SNK p<0.001, ANOVA
F1,8=460, p<0.001; Fig. 1). The toxin profiles were dominated by gonyautoxin (GTX) 1 and 4 (97.8±1.0% of total toxin content) with small amounts of GTX 2/3 present in all samples (2.2±1.0%). The relative contribution of GTX 1/4 to GTX 2/3 was not different between treatments on the first sampling occasion (tdf=4=0.96; p=0.39), but on the second sampling occasion, GTX 2/3 were slightly more common in controls (3.7±0.4%) compared to grazed treatments (1.9±0.1%; mean ±SD tdf=4=-11.7; p<0.001). Manuscript 2 89
Growth rates were similar between treatments over the course of the experiment
(tdf=4=0.28; p=0.79) and averaged 0.29 d-1. The density of Alexandrium cells approximately doubled over the experimental time, and the highest increase in cell numbers was observed over the first 24 hours (Fig. 2). Day 3 cell numbers differed by less than 4% between copepod-exposed and control cultures, indicating that only a small part of the Alexandrium cells was consumed. This is in agreement with theoretical considerations based on published C. typicus ingestion rates (Bonnet & Carlotti, 2001) indicating that four adult females should not consume more than 47000 cells, or 1.07% of the initial cell number, per day.
Nutrient concentration was still high at the end of the experiment (105±6.8 µM nitrate and 1,9±0.22 µM phosphate mean +-SD) indicating that the cultures were not nutrient limited.
Microarray data identified 14 genes as being significantly higher expressed in the copepod-exposed cultures (Table 1); no gene was identified as higher expressed in the control. 7 of these genes were significantly up-regulated only on Day 1, 3 only on Day
3, and 4 genes were identified as higher expressed in the copepod-exposed cultures at both sampling occasions.. The highest measured difference was the 11.4-fold higher expression of the sequence Amin_06b12r on Day 3. The same gene was also the one with the highest relative expression on Day 1, when it showed 9.9 fold greater expression in the treatment than in the control.
Two of the differentially expressed genes could be annotated (Table 1): Amin_08 10f, which was 3.8 fold higher expressed in the copepod-conatining cultures on Day 1, was identified as a peptidylprolyl isomerase based on sequence similarity searches against various nucleotide and protein sequence databases.Amin_56a03r, which showed 10 fold greater expression in copepod-challenged cultures on Day 3, contained a 139 amino Manuscript 2 90 acid domain recognised by Pfam (http://pfam.sanger.ac.uk/search) as indicative of a
NAD:arginine ADP-ribosyltransferase (ART); this was verified by BLAST hits at E- values below our threshold for BLAST-based annotation (e-value>10-4).
The other differentially expressed genes could only be annotated as “hypothetical proteins” due to a lack of similar sequences in the searched databases. For some of these sequences, prediction of the protein coding open reading frame (orf) remained uncertain as well, so signal peptides and transmembrane regions could not be predicted with confidence. BLAST searches against other dinoflagellate EST libraries revealed similar sequences (e-value < 10-5) for five of the differentially expressed genes (supplementary table).
Discussion
Inducible defences have been found in widely different organisms from prokaryotes
(Patterson & Bolis, 1997; Matz et al., 2004) to diatoms (Lürling & Van Donk, 2000), ciliates (e.g. Hammill et al., 2009) and various green, red and brown macroalgae (Toth
& Pavia, 2007), but investigations into the transcriptomal changes associated with induced chemical defences have largely been restricted to angiosperm model organisms
(Arimura et al., 2004; Reymond et al., 2004; De Vos et al., 2005). To our knowledge, this is the first study to link gene expression in a planktonic organism to its reaction to grazer presence.
Grazer induced elevation of PST formation in Alexandrium is mediated by distant chemoreception without the involvement of mechanical damage (Selander et al., 2006) and thus is a specific defensive response not confounded by wounding reactions.
Consequently, measured differences in gene expression have a higher probability of being involved in the induction of higher toxin cell quotas. Of the 14 genes found to be Manuscript 2 91 significantly up-regulated in copepod-exposed cultures, two have been identified in a previous study as considerably higher expressed in toxin-producing strains of A. minutum, as compared to a non toxin-producing strain (genes Amin_89d06r and
Amin_56a03r, expressed 9.88 and 51.01 folds higher in toxic strains, respectively
(Mansucript 1). As both studies identified only a small proportion of the tested genes as differentially expressed – 0.35% in this study and 4.82% in the strain comparison – this pattern suggests that these two sequences are good candidates for future studies of the regulation of PSP toxin content at the gene level.
The number of significantly differentially expressed genes in this study was lower than those identified between other physiological treatments in A. minutum, such as exponentially growing cultures under different salinities (Mansucript 2). While a slight strain-specific effect of the microarray probes cannot be ruled out, this further indicates that the differences in gene expression observed in this experiment are likely to be directly associated with either copepod recognition or induction of higher toxin content.
Typically for genomic studies in dinoflagellates (John et al., 2004; Jaeckisch et al.,
2008), most of the sequences identified as differentially expressed could not be annotated due to a profound lack of well-characterised similar sequences in all available databases.
Of the two annotatable genes, Amin_08c10f, which was up-regulated in the copepod- containing cultures on Day 1, was identifiable as a peptidylprolyl isomerase (PPIase).
This protein family catalyzes the cis-trans isomerization of proline imidic peptide bonds and thus accelerates protein folding (e.g. Pfam: pfam.sanger.ac.uk//family/Rotamase, uniprot: www.uniprot.org/keywords/?query=name:%22Rotamase%22). In some cases,
PPI-mediated peptidy- prolyl cis/trans isomerisation has been reported to act as a molecular switch mechanism between different confirmations of protein native states, which differ in ligand recognition or activity (Andreotti, 2003). Manuscript 2 92
The sequence Amin_56a03r that which showed 10fold greater expression in copepod- challenged high-toxin-containing cultures on Day 3, and which has also been associated with toxic strains (Mansucript 1), was identified as an NAD:arginine ADP- ribosyltransferase (ART). The ART domain catalyzes the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD+) to an arginine residue of an acceptor protein. This mono-ADP-ribosylation is a phylogenetically ancient mechanism of reversible posttrantlational modification found both in prokaryotes and eukaryotes
(reviewed in Hassa et al., 2006). It was originally identified as the pathogenic mechanism of several bacterial toxins, including cholera toxin, and is generally implicated in changing the activity of target proteins. The most well-studied eukaryotic
ART family members are a suite of mammalian extracellular free or membrane-bound enzymes acting on extracellular proteins (e.g. Okazaki et al., 1996), but arginine- specigic mono-ADP-ribosylation is also known from cytoplasmic and nuclear targets including histones. ADP-ribosylation of different proteins has been implicated in the control of proliferation, apoptose and differentiation of specific mammalian cell types
(Hassa et al., 2006).
The low proportion (0.35%) of 14 up-regulated genes out of 3983 sequences tested is reminiscent of the initial response of Arabidopsis towards insect attack. Reymond et al.
(2004) extrapolated the differentially expressed genes in Arabidopsis after 3-5 h of attack to 1.3% of the total transcriptome. Similar to our experiment, their study identified almost exclusively up-regulated transcripts. In contrast to this, the transcriptome of Nicotiana attenuata contained both up- and down-regulated genes after
24 h of attack by either mirids or hornworms (Voelckel & Baldwin, 2004). Although the
3 to 5 h response identified by Reymond et al. (2004) was almost identical between a specialist and a generalist insect, the 24 h Nicotiana responses against the mirids and the hornworms included more genes differentially expressed between attacking species than Manuscript 2 93 commonly regulated genes. This difference vanished within 5 days, leading to the conclusion that the plant’s first reaction is a rapid recognition response, which declines while the defense is mounted. A similar mechanism seems to exist in the response of A. minutum to C. typicus: While more genes are significantly induced after 1 day of exposure to the grazer, the physiological response of a considerably higher intracellular toxin content was measured after 3 days. This is consistent with the hypothesis of a short recognition and acclimation period, followed by a physiological switch to higher toxin accumulation.
It should be noted that Alexandrium spp produce several other bioactive compounds besides PST, e.g. spirolides (Cembella et al., 1999), and the hereto unidentified lytic compounds associated with the allelophathic effects in Alexandrium spp. (Tillmann &
John, 2002; Tillmann et al., 2008; Ma et al., 2009), and it is likely that there are additional bioactive secondary metabolites that we are not yet aware of and consequently will not detect when targeting only known substances. The microarray procedure on the other hand provides a non-biased tool to detect regulation of secondary or primary metabolism as long as this regulation is mediated through a change in transcriptional activity.
Conclusions
Overall, the results suggest that grazer-induced toxin formation may be a good starting point for investigations of genes involved in PST formation. Toxin levels can be manipulated with trace amounts of copepod cues, without the confounding effects of wounding (Selander et al., 2006; Selander, 2007). The results show that corresponding change in transcription is modest, suggesting that toxin induction is not accompanied by a massive amount of confounding factors, as is the case when toxicity is manipulated Manuscript 2 94 with e.g. phosphate limitation (Barreiro et al., 2006) or the use of different species
(Teegarden, 1999). A comparison of the gene expression response associated with grazer-induced elevated toxin formation with expression differences between toxin- producing and non-toxin-producing strains revealed two sequences with expression patterns suggesting involvement in the regulation of toxin content. The NAD:arginine
ADP-ribosyltransferase domain-containing sequence Amin_56a03r, which has been identified as having the highest relative expression of toxic vs non-toxic cultures, was also the gene with the highest relative expression in the high-toxicity-induced cultures on Day 3. We consider this sequence as a particularly strong candidate for involvement in the regulation of cellular toxicity.
Acknowledgements
The work was supported by the EU project ESTTAL (GOCE-CT2004-511154), Marine
Genomics Europe (GOCE-CT-2004-505403), the PACES research program of the
Alfred Wegener Institute as part of the Helmholtz Foundation initiative in Earth and
Environment, LIDPOP (IEF-237517), the Swedish Research Council Formas (contract
217-2007-955), by the Swedish Research Council (contract 621- 2007-5779), and by
MARICE, a strategic research platform in marine chemical ecology at the University of
Gothenburg.
References
ANDREOTTI, A. H. (2003). Native state proline isomerization: An intrinsic molecular switch. Biochemistry 42(32): 9515-9524.
ARIMURA, G., HUBER, D. P. W., BOHLMANN, J. (2004). Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1. The Plant Journal 37(4): 603-616.
BARREIRO, A., GUISANDE, C., FRANGÓPULOS, M., GONZÁLEZ-FERNÁNDEZ, A., MUÑOZ, S., PÉREZ, D., MAGADÁN, S., MANEIRO, I., RIVEIRO, I., IGLESIAS, P. (2006). Feeding strategies of Manuscript 2 95 the copepod Acartia clausi on single and mixed diets of toxic and non-toxic strains of the dinoflagellate Alexandrium minutum. Marine Ecology Progress Series 316: 115-125.
BEAUGRAND, G., LINDLEY, J. A., HELAOUET, P., BONNET, D. (2007). Macroecological study of Centropages typicus in the North Atlantic Ocean. Progress in Oceanography 72(2-3): 259-273.
BERGKVIST, J., SELANDER, E., PAVIA, H. (2008). Induction of toxin production in dinoflagellates: the grazer makes a difference. Oecologia 156(1): 147-154.
BONNET, D., CARLOTTI, F. (2001). Development and egg production in Centropages typicus (Copepoda: Calanoida) fed different food types: a laboratory study. Marine Ecology-Progress Series 224: 133-148.
CEMBELLA, A. D., LEWIS, N. I., QUILLIAM, M. A. (1999). Spirolide composition of micro-extracted pooled cells isolated from natural plankton assemblages and from cultures of the dinoflagellate Alexandrium ostenfeldii. Natural Toxins 7: 197-206.
CEMBELLA, A. D. (2003). Chemical ecology of eukaryotic microalgae in marine ecosystems. Phycologia 42(4): 420-447.
CEMBELLA, A. D., JOHN, U. (2006). Molecular physiology of toxin production and growth regulation in harmful algae. In Ecology of Harmful Algae (E. GRANÉLI , J. T. TURNER editors), pp. 215-227. Springer Berlin Heidelberg.
COLIN, S. P., DAM, H. G. (2002). Latitudinal differentiation in the effects of the toxic dinoflagellate Alexandrium spp. on the feeding and reproduction of populations of the copepod Acartia hudsonica. Harmful Algae 1(1): 113.
DE VOS, M., VAN OOSTEN, V. R., VAN POECKE, R. M. P., VAN PELT, J. A., POZO, M. J., MUELLER, M. J., BUCHALA, A. J., MÉTRAUX, J. P., VAN LOON, L. C., DICKE, M., PIETERSE, C. M. J. (2005). Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Molecular Plant-Microbe Interactions 18(9): 923-937.
ERDNER, D. L., ANDERSON, D. M. (2006). Global transcriptional profiling of the toxic dinoflagellate Alexandrium fundyense using massively parallel signature sequencing. BMC Genomics 7.
FLYNN, K., FRANCO, J. M., FERNÁNDEZ, P., REGUERA, B., ZAPATA, M., WOOD, G., FLYNN, K. J. (1994). Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress. Marine Ecology Progress Series 111: 99-109.
FRANCO, J., FERNÁNDEZ, P., REGUERA, B. (1994). Toxin profiles of natural populations and cultures of Alexandrium minutum Halim from Galician (Spain) coastal waters. Journal of Applied Phycology 6(3): 275.
HACKETT, J., SCHEETZ, T., YOON, H., SOARES, M., BONALDO, M., CASAVANT, T., BHATTACHARYA, D. (2005). Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6(1): 80.
HAMMILL, E., KRATINA, P., ANHOLT, B. (2009). Non-lethal presence of predators modifies morphology and movement rates in Euplotes. Hydrobiologia 621: 183-189.
HANSEN, G., DAUGBJERG, N., FRANCO, J. M. (2003). Morphology, toxin composition and LSU rDNA phylogeny of Alexandrium minutum (Dinophyceae) from Denmark, with some morphological observations on other European strains. Harmful Algae 2(4): 317-335.
HASSA, P. O., HAENNI, S. S., ELSER, M., HOTTIGER, M. O. (2006). Nuclear ADP-ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiology and Molecular Biology Reviews 70(3): 789-829.
HWANG, D. F., LU, Y. H. (2000). Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum. Toxicon 38(11): 1491-1503. Manuscript 2 96
JAECKISCH, N., GLÖCKNER, G., VOGEL, H., CEMBELLA, A., JOHN, U. (2008). Genomic characterization of the spirolide-producing dinoflagellate Alexandrium ostenfeldii with special emphasis on PKS genes. In Proceedings of 12th International Conference on Harmful Algae, Copenhagen, Denmark. 04.-08.09.2006 (Ø. MOESTRUP, G. DOUCETTE, H. ENEVOLDSEN editors)
JOHN, U., MOCK, T., VALENTIN, K., CEMBELLA, A. D., MEDLIN, L. K. (2004). Dinoflagellates come from outer space but haptophytes and diatoms do not. In Proceedings of Harmful Algae 2002, St. Pete Beach, Florida, USA. 21–25.10.2002 (K. A. STEIDINGER, J. H. LANDSBERG, C. R. TOMAS, G. A. VARGO editors)
KELLER, M. D., SELVIN, R. C., CLAUS, W., GUILLARD, R. R. L. (1987). Media for the culture of oceanic ultraphytoplankton. Journal of Phycology 23(4): 633-638.
KELLMANN, R., MICHALI, T. K., NEILAN, B. A. (2008a). Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers. Journal of Molecular Evolution 67(5): 526-538.
KELLMANN, R., MIHALI, T. K., JEON, Y. J., PICKFORD, R., POMATI, F., NEILAN, B. A. (2008b). Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Applied and Environmental Microbiology 74(13): 4044-4053.
LÜRLING, M., VAN DONK, E. (2000). Grazer-induced colony formation in Scenedesmus: are there costs to being colonial? Oikos 88(1): 111-118.
MA, H. Y., KROCK, B., TILLMANN, U., CEMBELLA, A. (2009). Preliminary characterization of extracellular allelochemicals of the toxic marine dinoflagellate Alexandrium tamarense using a Rhodomonas salina bioassay. Marine Drugs 7(4): 497-522.
MATZ, C., BERGFELD, T., RICE, S. A., KJELLEBERG, S. (2004). Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing. Environmental Microbiology 6(3): 218-226.
MIHALI, T., KELLMANN, R., NEILAN, B. (2009). Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. BMC Biochemistry 10(1): 8.
MOUSTAFA, A., LORAM, J. E., HACKETT, J. D., ANDERSON, D. M., PLUMLEY, F. G., BHATTACHARYA, D. (2009). Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS ONE 4(6): e5758.
OKAZAKI, I. J., KIM, H. J., MOSS, J. (1996). Cloning and characterization of a novel membrane- associated lymphocyte NAD:arginine ADP-ribosyltransferase. Journal of Biological Chemistry 271(36): 22052-22057.
PATTERSON, G. M. L., BOLIS, C. M. (1997). Fungal cellwall polysaccharides elicit an antifungal secondary metabolite (phytoalexin) in the cyanobacerium Scytonema ocellatum. Journal of Phycology 33(1): 54-60.
PLUMLEY, F. G. (1997). Marine algal toxins: Biochemistry, genetics and molecular biology. Limnology and Oceanography 42(5): 1252-1264.
REYMOND, P., BODENHAUSEN, N., VAN POECKE, R. M. P., KRISHNAMURTHY, V., DICKE, M., FARMER, E. E. (2004). A conserved transcript pattern in response to a specialist and a generalist herbivore. Plant Cell 16(11): 3132-3147.
SAEED, A. I., SHAROV, V., WHITE, J., LI, J., LIANG, W., BHAGABATI, N., BRAISTED, J., KLAPA, M., CURRIER, T., THIAGARAJAN, M., STURN, A., SNUFFIN, M., REZANTSEV, A., POPOV, D., RYLTSOV, A., KOSTUKOVICH, E., BORISOVSKY, I., LIU, Z., VINSAVICH, A., TRUSH, V., QUACKENBUSH, J. (2003). TM4: A free, open-source system for microarray data management and analysis. Biotechniques 34(2): 374-378. Manuscript 2 97
SELANDER, E., THOR, P., TOTH, G., PAVIA, H. (2006). Copepods induce paralytic shellfish toxin production in marine dinoflagellates. Proceedings of the Royal Society B: Biological Sciences 273(1594): 1673-1680.
SELANDER, E. (2007). Chemical ecology of paralytic shellfish toxin producing dinoflagellates. In Department of Marine Ecology, p. 128. Göteborg University, Strömstad.
STOREY, J. D. (2003). The positive false discovery rate: A Bayesian interpretation and the q-value. Annals Of Statistics 31(6): 2013-2035.
STRICKLAND, J. D. H., PARSONS, T. R. (1972). A practical handbook of seawater analysis, 2nd edition. Fisheries Research Board of Canada, Ottawa.
STUCKEN, K., JOHN, U., CEMBELLA, A., MURILLO, A. A., SOTO-LIEBE, K., FUENTES- VALDÉS, J. J., FRIEDEL, M., PLOMINSKY, A. M., VÁSQUEZ, M., GLÖCKNER, G. (2010). The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications. PLoS ONE 5(2): e9235.
TEEGARDEN, G. J. (1999). Copepod grazing selection and particle discrimination on the basis of PSP toxin content. Marine Ecology Progress Series 181: 163-176.
TEEGARDEN, G. J., CAMPBELL, R. G., ANSON, D. T., OUELLETT, A., WESTMAN, B. A., DURBIN, E. G. (2008). Copepod feeding response to varying Alexandrium spp. cellular toxicity and cell concentration among natural plankton samples. Harmful Algae 7(1): 33-44.
TILLMANN, U., JOHN, U. (2002). Toxic effects of Alexandrium spp. on heterotrophic dinoflagellates: an allelochemical defence mechanism independent of PSP-toxin content. Marine Ecology Progress Series 230: 47-58.
TILLMANN, U., ALPERMANN, T., JOHN, U., CEMBELLA, A. (2008). Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists. Harmful Algae 7(1): 52-64.
TOTH, G. B., PAVIA, H. (2007). Induced herbivore resistance in seaweeds: a meta-analysis. Journal of Ecology 95(3): 425-434.
TOUZET, N., FRANCO, J. M., RAINE, R. (2007). Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland. Toxicon 50(1): 106-119.
TURNER, J. T., TESTER, P. A. (1997). Toxic marine phytoplankton, zooplankton grazers, and pelagic food webs. Limnology and Oceanography 42(5): 1203-1214.
TUSHER, V. G., TIBSHIRANI, R., CHU, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proceedings of the National Academy of Sciences of the United States of America 98(9): 5116-5121.
URIBE, P., FUENTES, D., VALDÉS, J., SHMARYAHU, A., ZÚÑIGA, A., HOLMES, D., VALENZUELA, P. D. T. (2008). Preparation and analysis of an expressed sequence tag library from the toxic dinoflagellate Alexandrium catenella. Marine Biotechnology 10(6): 692-700.
VOELCKEL, C., BALDWIN, I. T. (2004). Herbivore-induced plant vaccination. Part II. Array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations. The Plant Journal 38(4): 650-663. Manuscript 2 98
Figures
Figure 1: Cell specific toxin content in Alexandrium minutum cultures exposed to copepod grazers (grey bars) and controls without copepods added (white bars), after one and three days of incubation. Bars show mean values of three replicates, error bars denote standard deviation of mean. Manuscript 2 99
Figure 2: Density of Alexandrium minutum cells over the experimental period. Start concentrations represents a single sample whereas all other symbols represents the mean value of three replicates. Error bars denote standard deviation of mean. Manuscript 2 100
Table
Table 1 Up-regulated genes. Sequences identified as significantly differentially expressed between copepod-exposed and control cultures. No sequence significantly higher expressed in the control cultures was detected. Fold changes as geometric mean of biological triplicates.
Fold change Fold change
Contig name exposed / exposed / Gene product
control (Day 1) control (Day 3)
Amin_06b12r 9.92 11.40 hypothetical protein
Amin_08b02r 5.36 9.26 hypothetical protein
Amin_07b11f 2.88 4.61 hypothetical protein
Amin_88h09r2 5.11 2.81 hypothetical protein
Amin_26k02r 5.10 hypothetical protein
Amin_78b09r 4.81 hypothetical protein
Amin_06h01f 4.45 hypothetical protein
Amin_08c10f 3.80 peptidylprolyl isomerase
Amin_86k10r 2.39 hypothetical protein
Amin_79e01f 2.18 hypothetical protein
Amin_89d06r 1.91 hypothetical protein
Amin_88f04r2 3.54 hypothetical protein
Amin_61f03r 3.99 hypothetical protein
Amin_56a03r 9.77 hypothetical protein Manuscript 2 101
Supplementary Table
Additional file: information on the genes up-regulated at Day 1 or Day 3 of copepod- induced PSP toxin production (see appendix)
Log2 values of fold change exposed / control (mean of triplicates), standard deviation of log2 values, fold change exposed / control (geometric mean of triplicates), annotation or lack of annotation confirmed manually. Geometric mean toxic strains / non-toxic strains
(Manuskript 1). With value of best BLAST hit against other dinoflagellate EST libraries, NCBI Transcriptome Shotgun Assembly (TSA) database accession numbers and miscellaneous sequence characteristics. Manuscript 3 102
7 Manuscript 3 Manuscript 3 103
Physiological and Gene Expression Responses to Salinity
Stress in Alexandrium minutum
I. Yang§, S. Beszteri, U. Tillmann, A. Cembella & U. John
All authors: Alfred Wegener Institute, Am Handelshafen 12, 27570 Bremerhaven,
Germany
§Corresponding author
Ines Yang
[email protected] Manuscript 3 104
Abstract
We investigated the effect of high- and low-salinity stress on the PSP-toxin-producing dinoflagellate Alexandrium minutum. The photosynthetic efficiency parameter Fv/Fm was affected throughout exponential growth but was most reduced in low salinity.
Chloroplast-related gene expression changed most in high salinity, whereas chloroplast efficiency was less affected, suggesting that these gene expression changes are linked to chloroplast acclimation. In exponential growth, 8.8% of genes tested were differentially expressed under salinity stress, but the amplitude of regulation was low (max 3.5 fold).
Salt stress acclimation seems to be regulated mainly post-transcriptionally, as 27% of the functionally annotated differentially expressed genes were involved in mRNA modification or translation-related processes. Consistent with other dinoflagellates, the high abundance of post-transcriptional and translational sequences identified in this microarray experiment suggests that this might be a general feature of dinoflagellate gene expression. Manuscript 3 105
Introduction
Alexandrium minutum is a bloom-forming toxic dinoflagellate occurring from northern
Europe and the Mediterranean to Asia, Australia and New Zealand. European strains typically produce gonyautoxins (GTX 1,2,3,4) associated with paralytic shellfish poisoning (PSP). This species preferably blooms in confined coastal habitats, such as harbours, estuaries or lagoons, where blooms are often initiated by input of nutrient-rich freshwater (Bravo et al., 2008). Recorded salinity preferences range from s = 15 (for strains isolated from brackish water, see Hwang & Lu, 2000) to s = 34-38 (Bravo et al.,
2008).
This study is part of the EU project Expressed Sequence Tags of Toxic ALgae
(ESTTAL; http://genome.imb-jena.de/ESTTAL/cgi-bin/Index.pl), in which we aim to examine the physiological and genetic basis for toxin production and bloom formation.
Here we focus on the physiology and gene expression of A. minutum at the strain- specific limits of growth tolerance at extreme salinities.
Methods
Following bacteria-reduction by gauze filtration and antibiotic treatment (Manuscript 1), triplicate cultures of A. minutum strain AL3T (origin: Gulf of Trieste, Italy) were grown at three different salinities. Control cultures were grown in modified K-medium based on sterile-filtered aged seawater (salinity s = 32.8 ± 0.2,; all salinities according to the practical salinity scale). High salinity medium was supplemented with 15 g l-1 NaCl to a final salinity of 49.7 ± 0.1; low-salinity medium (s = 11.9 ± 0.2) was prepared with ѿ aged seawater and Ҁ GHLRQLVHG ZDWHU &XOWXUHV ZHUH NHSW DW & DW D SKRWRQ IOX[ density of 200 µmol m-2 s-1 on a 16:8-h light:dark cycle. Cultures were monitored daily Manuscript 3 106 by pH measurements, microscopic cell counts and measurements of potential quantum efficiency (Fv/Fm) of Photosystem II by Pulse-Amplitude-Modulated (PAM)
-1 fluorometry. Specific growth rates were calculated: µ=(ln(Nt2)-ln(Nt1))(t2-t1) with
N=cells ml-1 and t=sampling time. Significance of physiological data was tested according to Student's t-test at p<0.05. Cells were harvested in mid- and late exponential growth and stationary phase.
PSP toxins were analyzed by liquid chromatography with fluorescence detection according to Krock et al. (2007). Allelochemical activity (as EC50) was determined by co-incubation of A. minutum with the cryptophyte Rhodomonas salina (Tillmann et al.,
2008).
Samples for gene expression analysis were taken during exponential growth, ca. 12 h after beginning of the light phase. RNA was extracted using the Sigma TriReagent protocol, followed by a modified Qiagen RNeasy cleanup (as in Manuscript 1). Total
RNA (500 ng sample-1) was amplified and labelled using an Agilent low-input linear amplification kit and hybridized to microarrays. Microarrays were scanned (Agilent
G2565AA scanner), and raw data were extracted and normalized by Agilent Feature
Extraction Software version 9.1.3.1. Output data were subject to SAM (Significance
Analysis of Microarrays; Tusher et al., 2001) as implemented in MeV. Probes with a
SAM-based q-value of <1% and a minimum fold change of 1.5 were considered to indicate differential expression of the corresponding genes.
Results and Discussion
Physiological Responses
After a highly variable lag-phase, specific growth rates in exponential phase and cell numbers at beginning stationary phase were not significantly different among Manuscript 3 107 treatments. We therefore consider that from the start of exponential phase the cultures were salt-stress acclimated.
The pH increased continuously during exponential phase, and during stationary phase was in the range found limiting for different dinoflagellate species (Siu et al., 1997;
Hansen et al., 2007). The values at the beginning of stationary phase and on the two consecutive days differed significantly (p=0.05) among salinities: The three-day mean in control medium was pH 9.07 ± 0.04, in low salinity pH 8.95 ± 0.04 and in high salinity 9.27 ± 0.05.
Dark-adapted values of FV/Fm showed major differences among treatments. Whereas control values increased during exponential growth from 0.61 ± 0.04 to 0.71 ± 0.01, high salinity values stayed at 0.57 ± 0.08 until late exponential phase, when they increased to 0.66 ± 0.06. Low salinity exponential phase Fv/Fm values (0.55 ± 0.06) were below the range of control culture stationary phase values (0.58 ± 0.04). These low values are interpreted as an indication of damage to Photosystem II, suggesting differences in chloroplast efficiency at different salinities.
Intracellular PSP toxin content was significantly lower in all salinity-stressed cultures than in control cultures, and significantly lower in high salinity than in low salinity
(Fig.1a). Our results correspond to those of Hwang and Lu (2000), whose A. minutum strain T1, which originated from an aquaculture pond in Taiwan, grew best at s = 15, showed reduced toxicity in s = 7.5 and reached even lower toxicity values at s = 25 and s = 30.
Irrespective of salinity, allelochemical activity against R. salina was higher in stationary phase than in mid-exponential phase, suggesting an accumulation of allelochemical compounds in the culture. Whereas the high salinity and control cultures showed overlapping confidence intervals of their EC50-values, the low salinity cultures were Manuscript 3 108 considerably less allelochemically active both in late exponential and in stationary phase (Fig.1b). This reinforces the finding by Tillmann & John (2002) that allelochemical activity in Alexandrium is not linked to intracellular PSP toxin content.
Here we showed that even within a strain, conditions that moderately affect PSP toxin content can drastically reduce allelochemical activity, whereas others leading to a more pronounced reduction in PSP toxin content do not significantly alter allelochemical potency.
Gene expression
The microarray analysis identified 320 genes as significantly differentially expressed in salinity-stressed when compared to control cultures, accounting for 8.8% of microarray probes. Of these, 23% (74 genes) could be functionally annotated (Fig. 2, table available on request), which is in the range expected for a dinoflagellate (John et al., 2004; Lidie et al., 2005; Jaeckisch et al., 2008). The average fold change of the genes identified as differentially expressed was 1.8, with a maximum value of 3.5.
In cultures under acute stress, general stress response genes should be expressed irrespective of the actual treatment. However, we observed no genes regulated in the same direction under both salinity-stress treatments. Instead, 12 genes were differentially expressed in both treatments but with opposite regulation; 8 of those were up-regulated in high- and down-regulated in low salinity, 4 others conversely. This response and the overall low gene expression differences may reflect the fact that acclimation was largely complete after lag phase.
Of the A. minutum genes differentially expressed in the salinity-stress treatments, 242 were up-or down-regulated in high salinity, and 90 changed their expression in low salinity. One of the most crucial steps of acclimation to salt stress is the re- establishment of ion homeostasis (Krell et al., 2008). Some of the ion transporters are Manuscript 3 109 likely still needed even after acclimation is achieved. We detected four differentially expressed sequences putatively involved in ion transport in our salt-stress acclimated samples, two of which were ion transporters up-regulated in high salinity.
Twenty of the functionally annotatable genes (27%) were involved in post- transcriptional or translational processes. Most were up-regulated in high salinity (7 genes) or down-regulated in low salinity (8 genes). Among these were genes involved in pre-mRNA processing, ribosome biogenesis and translation (table available on request). In high salinity, we also found three differentially expressed genes for protein modification. Overall these data suggest an increase in mRNA processing, translation and protein modification in high salinity and a decrease in low salinity. The high proportion of sequences involved in post-transcriptional and translational processes among the differentially abundant mRNAs suggests that an important part of gene regulation in A. minutum occurs during these stages. This is in accordance with the finding that 33% of diurnally controlled identifiable genes in Karenia brevis were involved in posttransciptional processing/turnover or protein processing (Van Dolah et al., 2007).
Genes implicated in chloroplast metabolism were mainly affected by high salinity. We identified 2 down-regulated cytochrome b6 genes, whereas 8 other essential chloroplast- related genes were up-regulated. These included 4 light-harvesting complex proteins, a photosystem reaction centre and 2 different ATP synthase subunits. The only putative chloroplast gene differentially expressed in low salinity was a PPR repeat protein, which was down-regulated. This conspicuous reaction of chloroplast-associated genes to high salinity is similar to the results showing restructuring of the light harvesting complexes in the diatom Fragilariopsis cylindrus (Krell et al., 2008) in high-salinity shocked cultures. In contrast to the situation in high salinity, where major changes in Manuscript 3 110 chloroplast-associated gene expression are combined with a moderate reduction in
Fv/Fm values, the major drop in chloroplast efficiency seen in low salinity is not directly reflected in the expression of known chloroplast genes. However, the down- regulation of genes associated with RNA processing and translation in low salinity hints to an overall reduction in metabolic activity.
Protein-kinases probably mediate most of the differences among A. minutum cultures grown in different salinities, either via signal transduction or by direct phosphorylation of target enzymes. A total of 9 sequences identified as differentially expressed were annotated as proteins involved in signal transduction pathways. These included 8 protein kinases of different specificities. A putative calcium-dependent protein kinase was up-regulated in high salinity and down-regulated in low salinity and thus might be a candidate for the regulation of salinity-specific responses.
From this dataset we cannot draw any direct conclusion on which genes might be involved in toxicity or allelochemical activity. The cyanobacterial PSP toxin gene cluster found by Kellmann et al. (2008) has no identifiable homologues in our EST database, nor in the published databases of other PSP-producing Alexandrium species
(Hackett et al., 2005; Uribe et al., 2008), which might be due either to necessarily incomplete coverage of the dinoflagellate transcriptomes or to the huge phylogenetic distance between cyanobacteria and dinoflagellates. However, the data presented in this paper can be used in comparisons with other physiological treatments to find genes that are expressed in correlation with the production of PSP toxins or allelochemical substances. Manuscript 3 111
Acknowledgements
This study was financed by EU-project ESTTAL (GOCE-CT2004-511154), Marine
Genomics Europe (GOCE-CT-2004-505403) and the PACES research program of the
Alfred Wegener Institute as part of the Helmholtz Foundation initiative in Earth and
Environment. Manuscript 3 112
References
BRAVO, I., VILA, M., MASÓ, M., FIGUEROA, R. I., RAMILO, I. (2008). Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches. Harmful Algae 7(4): 515.
HACKETT, J., SCHEETZ, T., YOON, H., SOARES, M., BONALDO, M., CASAVANT, T., BHATTACHARYA, D. (2005). Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6(1): 80.
HANSEN, P. J., LUNDHOLM, N., ROST, B. (2007). Growth limitation in marine red-tide dinoflagellates: effects of pH versus inorganic carbon availability. Marine Ecology-Progress Series 334: 63-71.
HWANG, D. F., LU, Y. H. (2000). Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum. Toxicon 38(11): 1491-1503.
JAECKISCH, N., GLÖCKNER, G., VOGEL, H., CEMBELLA, A., JOHN, U. (2008). Genomic characterization of the spirolide-producing dinoflagellate Alexandrium ostenfeldii with special emphasis on PKS genes. In Proceedings of 12th International Conference on Harmful Algae, Copenhagen, Denmark. 04.-08.09.2006 (Ø. MOESTRUP, G. DOUCETTE, H. ENEVOLDSEN editors)
JOHN, U., MOCK, T., VALENTIN, K., CEMBELLA, A. D., MEDLIN, L. K. (2004). Dinoflagellates come from outer space but haptophytes and diatoms do not. In Proceedings of Harmful Algae 2002, St. Pete Beach, Florida, USA. 21–25.10.2002 (K. A. STEIDINGER, J. H. LANDSBERG, C. R. TOMAS, G. A. VARGO editors)
KELLMANN, R., MICHALI, T. K., NEILAN, B. A. (2008). Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers. Journal of Molecular Evolution 67(5): 526-538.
KRELL, A., BESZTERI, B., DIECKMANN, G., GLÖCKNER, G., VALENTIN, K., MOCK, T. (2008). A new class of ice-binding proteins discovered in a salt-stress-induced cDNA library of the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae). European Journal of Phycology 43(4): 423 - 433.
KROCK, B., SEGUEL, C. G., CEMBELLA, A. D. (2007). Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry. Harmful Algae 6(5): 734-744.
LIDIE, K. B., RYAN, J. C., BARBIER, M., VAN DOLAH, F. M. (2005). Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of DNA microarray. Marine Biotechnology 7(5): 481-493.
SIU, G., YOUNG, M., CHAN, D. (1997). Environmental and nutritional factors which regulate population dynamics and toxin production in the dinoflagellate Alexandrium catenella. Hydrobiologia 352(1): 117.
TILLMANN, U., JOHN, U. (2002). Toxic effects of Alexandrium spp. on heterotrophic dinoflagellates: an allelochemical defence mechanism independent of PSP-toxin content. Marine Ecology Progress Series 230: 47-58.
TILLMANN, U., ALPERMANN, T., JOHN, U., CEMBELLA, A. (2008). Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists. Harmful Algae 7(1): 52-64.
TUSHER, V. G., TIBSHIRANI, R., CHU, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proceedings of the National Academy of Sciences of the United States of America 98(9): 5116-5121. Manuscript 3 113
URIBE, P., FUENTES, D., VALDÉS, J., SHMARYAHU, A., ZÚÑIGA, A., HOLMES, D., VALENZUELA, P. D. T. (2008). Preparation and analysis of an expressed sequence tag library from the toxic dinoflagellate Alexandrium catenella. Marine Biotechnology 10(6): 692-700.
VAN DOLAH, F. M., LIDIE, K. B., MOREY, J. S., BRUNELLE, S. A., RYAN, J. C., MONROE, E. A., HAYNES, B. L. (2007). Microarray analysis of diurnal- and circadian-regulated genes in the Florida red- tide dinoflagellate Karenia brevis (Dinophyceae). Journal of Phycology 43(4): 741-752. Manuscript 3 114
Figures
Figure 1a Intracellular toxin content. Mean values of triplicate cultures. Bars = SD
(n=3). 1b Allelochemical activity against R. salina. Bars = 95% confidence intervals, low salinity, s = 11.9 ± 0.2; control, s = 32.8 ± 0.2; high salinity, s = 49.7 ± 0.1 115
Figure 2 Functional categories of genes identified as differentially expressed between salinities. Positive range = up-regulated, negative range = down-regulated. Manuscript 4 116
8 Manuscript 4 Manuscript 4 117
Growth- and nutrient- dependent gene expression in
Alexandrium minutum
Ines Yang1,2, Sára Beszteri1, Urban Tillmann1, Allan Cembella1,Uwe John1
1 Alfred-Wegener-Institut für Polar- und Meeresforschung, Am Handelshafen 12, 27570
Bremerhaven, Germany
2 School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK Manuscript 4 118
Abstract
The toxigenic marine dinoflagellate Alexandrium minutum forms toxic blooms causing paralytic shellfish poisoning (PSP), primarily in coastal waters, throughout the world.
We examined effects on physiology and gene expression patterns associated with growth and nutrient starvation in a toxic strain of A. minutum. Bloom-relevant factors, including growth rate, intracellular toxin content, allelochemical activity and nutrient status were investigated in A. minutum cultures grown under different environmental regimes. Allelochemical potency of A. minutum increased with culture age but was independent of nutrient status.
The phenotypic data were integrated and compared with gene expression in cell samples taken at selected points along the growth curve. We observed 489 genes consistently differentially expressed between exponentially growing and growth-limited cultures. The expression pattern of stationary-phase cultures was characterised by conspicuous down-regulation of translation-associated genes, up-regulation of sequences involved in intracellular signalling and some indications of increased activity of selfish genetic elements such as transposons. Treatment-specific patterns included five genes regulated in parallel in all nutrient-limited cultures. The conspicuous decrease in photosynthetic performance identified in N-starved cultures was paralleled by down-regulation of chloroplast-associated genes.
The particular gene expression patterns we identified as specifically linked with exponential growth, cessation of growth or nutrient limitation may be suitable biomarkers for indicating the beginning of growth limitation in field- or mesocosm studies. Manuscript 4 119
Introduction
Dinoflagellates are ubiquitous protists and key components of marine and freshwater food webs worldwide. In many marine systems, chloroplast-containing dinoflagellates are among the most important biomass producers (Yallop, 2001; Anderson et al., 2008;
Thompson et al., 2008). Many dinoflagellate species can form dense blooms. To a much larger extent than those of other planktonic groups, dinoflagellate blooms often pose serious health and ecosystem threats through the production of noxious, toxic or other ecosystem-disruptive substances.
Alexandrium minutum is a widely distributed toxic dinoflagellate that tends to form toxic blooms associated with paralytic shellfish poisoning (PSP) in warm temperate coastal regions worldwide, from the Mediterranean (Bravo et al., 2008) to subtropical
Asia (Hwang & Lu, 2000), and New Zealand (Chang et al., 1997). Alexandrium minutum can grow under a relatively wide range of temperatures and salinities, and under low-turbulent conditions bloom development seems to be largely dependent on nutrient availability (Vila et al., 2005; Bravo et al., 2008). This implies that under non- bloom conditions A. minutum growth has a tendency to be nutrient-limited, therefore data on the physiological and gene expression differences between exponentially growing and nutrient-limited cultures are of high ecological significance. As the different growth stages in laboratory batch cultures correspond to profound physiological differences that develop over a time-scale of several cell cycles (John &
Flynn, 2000), this species is also a convenient model to examine transcriptional regulation associated with acclimation of a dinoflagellate over physiologically relevant time-scales. Manuscript 4 120
Dinoflagellates are often considered to exhibit poor nutrient uptake efficiency and relatively slow growth rates when compared with other phytoplankton, such as diatoms
(Smayda, 1997), but this may be compensated by the typical dinoflagellate traits of circadian nutrient-retrieval migrations, high prevalence of mixotrophy, and production of allelochemicals and toxins targeted against interspecific competitors and predators
(Smayda, 1997; Cembella, 2003). Some of these substances are active against other protists (Tillmann & John, 2002), while others have harmful or toxic effects on other organisms, including humans.
In order to better understand mechanisms of population dynamics and bloom formation in dinoflagellates, more knowledge of the intrinsic regulation of growth, nutrient uptake and starvation responses, as well as the biosynthesis and regulation of toxins and allelochemical substances, is required. A combination of chemical characterisation, physiological experimentation and gene expression comparisons under a variety of environmental regimes seems most promising (Cembella & John, 2006). The chemistry of dinoflagellate toxins is well known, and apart from a few newly discovered species
(e.g., Tillmann et al., 2009), physiological responses related to growth and toxin production in toxin-producing dinoflagellates is often well studied (Flynn et al., 1994;
Chang & McClean, 1997; Yamamoto & Tarutani, 1999; Hwang & Lu, 2000; Leong et al., 2004; Touzet et al., 2007).
Genomic studies on dinoflagellates, however, are complicated by profound doubts as to what extent methods and concepts developed in other model organisms, including protists, are applicable to dinoflagellates (Moreno Díaz de la Espina et al., 2005;
Bachvaroff & Place, 2008; Monroe & Van Dolah, 2008). Dinoflagellates arguably contain the most unusual eukaryotic genetic machinery known. Their huge genomes
(LaJeunesse et al., 2005) comprise both major proportions of apparently random, non- Manuscript 4 121 repetitive DNA with very little recognizable gene content (McEwan et al., 2008) and unusually high numbers of transcribed genes (Moustafa et al., 2010). For example,
Alexandrium tamarense, with about three times the nuclear DNA content of A. minutum, was shown to contain about 40,000 transcribed genes occurring in complex families (LaJeunesse et al., 2005). Dinoflagellate chromosomes are permanently condensed into a liquid crystal state (Livolant & Bouligand, 1978; Moreno Díaz de la
Espina et al., 2005), and transcription as well as most of the coding sequences seem to be restricted to DNA filaments protruding into the nucleoplasm (Anderson et al., 1992).
Partly owing to these genomic peculiarities, fundamental aspects about the regulation of dinoflagellate gene expression are currently under debate.
Contradictory evidence exists regarding the extent of gene regulation on the transcriptomic level. Both regulation of mRNA abundances (Taroncher-Oldenburg &
Anderson, 2000; Okamoto & Hastings, 2003; Hosoi-Tanabe et al., 2005; Toulza et al.,
2010) and a high prevalence of translational regulation (Rossini et al., 2003; Lidie,
2007; Lapointe & Morse, 2008) have been reported in dinoflagellates. The discovery of spliced-leader trans-splicing (Lidie & Van Dolah, 2007; Zhang et al., 2007; Slamovits
& Keeling, 2008) and of single-domain transcripts apparently derived from multi- domain genes led to the suggestion of trypanosome-like mechanisms of spliced-leader- associated constitutive translational gene regulation in dinoflagellates (Monroe & Van
Dolah, 2008). In analogy to trypanosomes, highly expressed dinoflagellate genes were proposed to be constitutively transcribed and regulated during mRNA processing in a mechanism involving trans-splicing (Bachvaroff & Place, 2008). This model would predict transcriptional regulation to be restricted to low-copy genes mostly lacking spliced leader sequences. However, this has been challenged by the discovery of spliced leader sequences in the 5’-regions of the genes postulated to lack them (Zhang & Lin,
2009). Manuscript 4 122
We investigated growth-related processes in batch cultures of A. minutum in exponential versus stationary growth phase and under nutrient starvation, to gain a deeper understanding of the physiological and transcriptomic processes associated with bloom formation and development. In addition to determining the phenotypic effect on toxin content and allelochemical activity, we compared the transcriptional response of exponentially growing and growth-limited batch cultures under different growth regimes along the growth curve. At three characteristic points of the culture cycle, we determined allelochemical activity, intracellular toxin content and intracellular and extracellular nutrient status. By means of DNA microarrays, we compared gene expression differences among cultures in exponential growth (control), at the transition to stationary phase, and several days after onset of stationary phase. Cross-comparison of the resulting patterns of differential gene expression enabled us to propose characteristic expression patterns associated with specific physiological phenomena.
Methods
Strain and culture conditions
Alexandrium minutum strain AL3T (origin: Gulf of Trieste, Italy) was grown at 20°C at a photon flux density of 200 µmol m-2 s-1 on a 16:8 h light:dark cycle. Stock cultures were kept in modified K-medium consisting of aged seawater (salinity ca. 32 practical
-1 - -1 + -1 3- salinity units) enriched with 440 µmol L NO3 , 36 µmol L NH4 , 25 µmol L PO4 ,
-1 2- -1 10 nmol L SeO3 , 1000 µmol L Trizma-Base (pH 8.3), K trace metal solution and f/2 vitamin solution (Keller et al., 1987). Preparatory cultures were filtered over a 10 µm gauze, washed with sterile-filtered seawater to reduce bacterial load and grown under antibiotic treatment (50 µg mL-1 ampicillin, 33 µg mL-1 gentamicin, 10 µg mL-1 ciprofloxacin, 1.13 µg mL-1 chloramphenicol and 0.025 µg mL -1 streptomycin sulfate) Manuscript 4 123 for 13 days, during which they were kept in exponential growth phase by repeated sub- culturing. Only starter cultures in which no bacteria could be detected by Acridine orange staining (Hobbie et al., 1977) followed by fluorescence microscopy were used to inoculate the experimental treatments.
Experimental cultures were grown in 5 L Duran bottles (Schott AG, Mainz, Germany) under constant gentle aeration and sampled with a sterile tube-vacuum system as described in Eschbach et al. (2005). Control cultures were grown in complete K- medium as defined above; for P- or N-limited cultures, the phosphate source or the nitrate and ammonium sources were omitted, respectively, from the medium.
Sampling and daily measurements
Cultures were monitored daily by pH measurements, microscopic cell counts and measurements of potential quantum efficiency (Fv/Fm) of Photosystem II. The Fv/Fm values were determined by Pulse-Amplitude-Modulated (PAM) fluorometry using a
Xenon-PAM-Fluorometer (WALZ GmbH, Effeltrich, Germany) after 15 ±5 min of dark incubation, following the method detailed by Mock and Hoch (2005). Specific growth
-1 -1 rates were calculated: µ = (ln(Nt2)-ln(Nt1))(t2-t1) with N = cells mL and t = sampling day. Stationary phase was defined as the growth phase where µ < 0.1 d-1. Samples for nutrient measurements, allelochemical assays, PSP toxin measurements, and RNA extraction were taken on Days 4 and 5 for all cultures and two to three days after each treatment triplicate had entered stationary growth phase. On the last treatment-specific sampling date, aliquots of each culture were transferred into 50 mL Erlenmeyer flasks to serve as run-on cultures for further monitoring of cell growth. In order to confirm nutrient limitation, two aliquots per nutrient-limited culture were taken, one of which was supplemented with the missing nutrient. Manuscript 4 124
Nutrient analysis
Filtered medium samples for dissolved nutrient analysis were preserved by adding 3 µL
3.5% (w/w) HgCl2 per mL sample and stored at 4 °C until analysis. Dissolved nutrients were analysed by continuous-flow analysis with photometric detection (AA3 Systems,
Seal GmbH, Norderstedt, Germany). For total dissolved phosphorus and nitrogen, the analysis was preceded by digestion with peroxodisulphate in an autoclave.
Samples for particulate nutrient analysis were filtered on pre-combusted glass fibre
GF/F filters (Whatmann, Omnilab, Bremen, Germany) and stored at -20°C. Filters for
C/N-measurements were dried at 60°C and encapsulated into chloroform-washed tin containers. Samples were analysed on an NA 1500 C/N Analyzer (Carlo Erba
Instrumentazione, Milan, Italy). Particulate phosphate was measured photometrically by continuous-flow analysis with photometric detection (AA3 Systems, Seal GmbH,
Norderstedt, Germany) after digestion with peroxide and sulphuric acid (Kattner &
Brockmann, 1980). Mean C/N values were calculated from the C/N measurements for individual filters; C/P and N/P values were determined from the average of all possible pairs of measurements for each culture at a given sampling point.
Toxin analysis
PSP toxins were extracted and prepared for analysis following the method described in
Krock et al. (2007) (see Manuscript 1). Briefly, cells were harvested by centrifugation
(3,000 x g, 4 °C). Pellets were suspended in 0.03 N acetic acid and homogenized in
FastPrep tubes containing 0.9 g of lysing matrix D with a Bio101 FastPrep instrument
(Thermo Savant, Illkirch, France) at maximum speed (6.5) for 45 s. Cell debris was removed by centrifugation at 16,100 x g at 4 °C for 15 min. The supernatant was filtered through a 0.45 ȝm pore-size spin-filter (Millipore Ultrafree, Eschborn,
Germany) by centrifugation for 30 s at 800 x g. PSP toxins were separated by ion-pair Manuscript 4 125 liquid chromatography and detected fluorometrically after post-column derivatization
(LC-FD) as described in Krock et al. (2007).
Determination of allelochemical activity
Allelochemical activity was determined by co-incubation of A. minutum cells with intact cultured cells of the cryptophyte Rhodomonas salina (Tillmann et al., 2008).
Briefly, different concentrations of A. minutum cells (in biological triplicates) were incubated with R. salina cells for 24 h in 20 ml glass vials in darkness. Incubations were stopped by addition of Lugol’s iodine solution and numbers of intact R. salina cells were counted using an inverted microscope (Zeiss, Jena, Germany) at 200 - 400X magnification The Alexandrium cell concentrations yielding a 50% decline in intact R. salina cells (EC50) were estimated by fitting the following equation to the cell count data using the non-linear fit procedure of Statistica (Statsoft, Germany):
h Nfinal = Ncontrol / (1 + (x / log EC50) )
with Nfinal = R. salina cell concentration after incubation with A. minutum, Ncontrol = R. salina cell concentration after incubation without A. minutum, x = log-transformed cell concentration of A. minutum, and the fit parameters log EC50 and h. Results are expressed as EC50 including 95% confidence intervals. Because of the high number of data points required to fit the equation, the data from all three replicate cultures were combined to calculate one EC50 value per treatment and time-point.
RNA extraction and microarray experiments
RNA extraction and microarray hybridisation were carried out as described in
Manuscript 1. Cells were harvested by filtration upon an 8 µm pore-sized filter
(TETP04700, Millipore Schwalbach, Germany) and rinsed with filter-sterilized seawater. Filters were quick-frozen in liquid nitrogen and later thawed by rinsing with Manuscript 4 126 heated (60°C) TriReagent (Sigma-Aldrich, Steinheim, Germany). RNA was extracted according to the TriReagent protocol, following cell lysis by 10 min incubation at 60 °C in TriReagent, aided by repeated vortex mixing with glass beads included in the sample tube. Briefly, after addition of 200 mL chloroform per mL TriReagent, samples were centrifuged for 15 min at 12,000 x g at 4 °C. The aqueous phase was mixed with an equal volume of isopropanol and incubated at -20 °C for at least 10 minutes. An RNA pellet was obtained by centrifugation at 12,000 x g for 10 min at 4°C. The pellet was washed by addition of 75% ethanol, followed by another centrifugation step. After removal of the ethanol, the pellet was dried until hyaline and then dissolved in 100 mL
RNAse-free water (Qiagen, Hilden, Germany). RNA cleanup and DNA digestion followed the protocol supplied with the Qiagen RNeasy kit: RNA samples were mixed with 350 mLELQGLQJEXIIHU5/7FRQWDLQLQJȕ-mercaptoethanol. After mixing with
250 mL ethanol, samples were applied to an RNeasy column (Qiagen) containing a silica membrane. Columns were washed by 1 min incubation with 700 mL RW1 followed by centrifugation before 10 mL DNase I mixed with 70 mL Buffer RDD (both
Qiagen) were applied for 15 min. To interrupt DNase digestion, columns were washed with 700 mL RW1. Samples were incubated for 1 min in Buffer RPE (Qiagen), centrifuged, and washed again with the same buffer. After 2 min centrifugation and another 1 min high-speed centrifugation in a new collection tube, RNA was eluted with
40 mL RNase-free water (Qiagen). To increase final RNA concentration, the flow- through was applied to the membrane a second time. When necessary, an additional cleanup and concentration step using Qiagen MinElute or Microcon Ultracel YM-30 columns was applied. RNA purity and quantity were determined with a NanoDrop
ND-1000 Spectrophotometer V3.1.0 (PeqLab, Erlangen, Germany), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen,
Germany). Manuscript 4 127
Total RNA (500 ng sample-1) was amplified and labelled using an low-input linear amplification kit (Agilent, Waldbronn, Germany), following the Agilent protocol for synthesis of Cy3- and Cy5-labelled cRNA and microarray hybridisation. Agilent custom-made microarrays were based on the oligonucleotide probe set previously developed (Manuscript 1). Day 5 and stationary-phase samples from all treatments were hybridised against control-treatment Day 4 samples. Microarrays were scanned on an
Agilent G2565AA scanner, and raw data was extracted with the Agilent Feature
Extraction Software version 9.1.3.1 (FE). Array quality was monitored using the
Agilent QC Tool (v1.0) with the metric set GE2_QCMT_Feb07.
Pre-processed data were analyzed using SAM (Significance Analysis of Microarrays
Tusher et al., 2001) as implemented in MeV 4.0 (Saeed et al., 2006), and SAM-based q- values (Storey, 2003) were calculated. The SAM one-class option served to compare each treatment to the control treatment Day 4 samples hybridised on the same arrays.
Probes with a q-value of <1% were considered to indicate differential expression of the corresponding genes if the mean fold change of the sample triplicate was at least 1.5. As all treatments had been hybridised against the same control samples, two-class SAM was applied to directly compare between treatments on Day 5 respectively in stationary phase. After identification of probes recognised as differentially expressed in several comparisons, the corresponding contig sequences in the A. minutum EST library were manually annotated.
Statistical analysis
Except for allelochemical activity (see above), physiological values are reported as the mean of biological triplicates with the associated standard deviation. Where not otherwise stated, significance of physiological data was tested according to the Student's t-test at p<0.05 (t-test). Normality was assessed by the Shapiro-Wilk-test as Manuscript 4 128 implemented in R and variances were compared by Fisher’s F-test. Significance of physiological data for which Fisher’s F-test indicated unequal variances was tested according to the Welch's t-test for unequal variances at p<0.05 (Welch test).
Microarray-based expression values are given as the geometric mean of three microarray measurements based on biological triplicates.
Results
Growth and physiological parameters
The nutrient-limited and control cultures displayed similar growth patterns during the early growth stages (Fig.1a). None of the cultures exhibited a pronounced lag phase, and mean exponential-phase growth rates were not significantly different between samples and among treatments (two-tailed t-test, p=0.1; Fig. 1b). Cell concentrations increased exponentially until Day 4, after which growth rate began to decrease under all treatments. Stationary phase was reached at Day 6 in the N-limited culture, Day 7 in the
P-limited culture and Day 8 in the control treatment (see Table 1 for stationary-phase cell counts, pH and nutrient ratios). Follow-up cultures after the stationary-phase harvesting point confirmed the specific nutrient limitation: aliquots of both the P- and the N-restricted treatments resumed growth after addition of the limiting nutrient, but those without added nutrients remained in stationary phase (Fig. 1c). Follow-up cultures of the control treatment, presumably containing sufficient extracellular and/or intracellular residual N- and P-nutrients, resumed growth as well. The pH values increased with increasing cell concentration; the highest values were reached in the control treatment just before stationary phase (Figs. 1b and 2).
Throughout the experiment, the nutrient-limited culture media contained greatly reduced amounts of the limiting nutrient (Fig. 3a), which was reflected in the Manuscript 4 129 intracellular nutrient levels and ratios (Fig. 3b, c). The stationary-phase control cultures had depleted the dissolved phosphate to levels similar to the exponentially growing P- limited culture by the time of the last harvesting, but intracellular P levels and P/C ratios were not significant different from those of N-limited cultures (Fig. 3c and Table 1).
The potential quantum efficiency of Photosystem II (PSII), measured as Fv/Fm, increased with increasing cell concentrations in exponentially growing control and P- limited culture (Fig. 4) and slowly decreased during stationary phase. In N-limited cultures, Fv/Fm did not change significantly during exponential phase (one-way
ANOVA Fv/Fm versus time) but decreased rapidly during stationary phase.
The changes in intracellular PSP toxin content along the growth curve were strongly treatment-dependent (Fig. 5a; significance tested according to Student's t-test at p<0.05). At the first two sampling points, toxin content per cell was not significantly different between nutrient-replete control and P-limitation conditions. Stationary-phase cellular toxin concentrations significantly declined in control cultures. In P-limited cultures, toxin per cell was significantly elevated both with respect to values from control or N-limited cultures and to values at the second sampling point from all treatments. Intracellular toxin content in the N-limited culture was significantly lower than in the other treatments at all sampling time points but did not change significantly over the culture cycle (t-test repectively Welch test at p<0.05).
Allelochemical activity against Rhodomonas salina followed the same trend in all cultures. Whereas the P-limited culture was much less allelochemically active at the first two sampling points, as indicated by much higher EC50 values, allelochemical activity increased with culture age in all three treatments (Fig. 5b). Manuscript 4 130
Gene expression
Out of 4,298 A. minutum sequences represented in the database, 1,781 (41%) were identified as differentially expressed between exponential-phase control samples and at least one of the treatments at the second or third sampling time-point (Table 2 a,b).
Among these genes, 1,025 were differentially expressed between one or both of the nutrient-limited treatments and the exponentially growing control.
The set of 1,565 genes up-regulated in the stationary phase compared to exponentially growing control cultures contained two carbonic anhydrase (CA) sequences. A dinoflagellate-type extracellular delta-CA, Amin_85n03r, was expressed 1.65-fold higher in the stationary phase control. This sequence was also 2.09-fold up-regulated in the stationary-phase N-limited cultures when compared to exponentially growing controls. The second sequence Amin_77b06f is an intracellular CA similar to a sequence known in the pennate diatom Phaeodactylum tricornutum. This gene was
2.12- fold up-regulated in the stationary-phase control relative to the exponentially growing control. For both Amin_85n03r and Amin_77b06f, expression differences between all other tested pairs of growth conditions remained non-significant.
In searching for consistent patterns of up- and down-regulation among datasets, we identified 554 sequences repeatedly associated with one of the tested physiological regimes (Table 3). The expression of 489 genes was linked to the difference between exponential growth and all tested growth-limiting conditions for A. minutum cultures.
Table 4 depicts a selection of these genes for which a function was assignable.
In stationary-phase control cultures 8 genes were down-regulated relative to exponential-phase controls and relative to both nutrient-limited treatments (Table 5); these were identified as characteristic for the stationary phase in control cultures. Manuscript 4 131
In both the comparisons with exponential- and stationary-phase cultures (Tables 3 and
6), 87 genes were associated with N- or P-limitation. Among these genes, 5 were regulated in parallel under all nutrient-limited regimes (Table 3, supplementary Table
1), but none of them could be annotated to function.
Analysis of the frequency of complete spliced leader (SL) sequences in the set of differentially expressed genes in comparison with the whole underlying EST library revealed no apparent pattern. Examination of the associated EST contigs showed that
4.4% of the genes differentially expressed between one of the treatments and the exponential-phase control samples contained a complete SL sequence. The same was true for 5.8% of the sequences that could be linked to physiological conditions. Both percentage values are similar to the 4.7% SL-containing sequences in the whole library.
Discussion
Nutrient limitation
For both nutrient-limited and control cultures, nutrient quotas and ratios were in the range of values previously published for A. minutum in laboratory experiments (Flynn et al., 1994; Maguer et al., 2007), and also agreed with those reported for other
Alexandrium species (John & Flynn, 2000; Juhl, 2005). Molar nutrient ratios (C:N, C:P,
N:P) in the control cultures were at the lower range of the reported values for nutrient- replete cultures of various marine microalgal species (Geider & La Roche, 2002).
Control culture values were also lower (Fig. 3c) than the canonical Redfield ratios
(Redfield, 1958) of mol C:N:P = 106:16:1 considered to represent balanced growth conditions in natural populations. Similar to other Alexandrium species, A. minutum is known to be a specialist for intracellular storage of P (Labry et al., 2008), and to a lesser extent of N (Flynn et al., 1996; Maguer et al., 2007), during nutrient pulses. The Manuscript 4 132 nutrients stored during the periods of “luxury consumption” can later be mobilised for growth when extracellular nutrients are depleted. Laboratory cultures are typically grown on “excess” inorganic N and P often at high external N:P ratios. Our A. minutum control cultures were inoculated into K-PHGLXPDWȝPRO/-1 total inorganic N and
-1 3- 25 ȝPRO / PO4 , apparently triggering significant intracellular nutrient storage and correspondingly reduced nutrient ratios.
As expected, in the nutrient-limited cultures, the intracellular amount of the limiting nutrient decreased both in absolute and in relative terms (Fig. 3b,c), indicating that acclimation to these conditions involves major changes in intracellular biochemistry.
The N:P ratio of 44.6+/-11.8 reached in the P-limited cultures were somewhat lower than those typically attained by a variety of marine microalgae under P limitation
(Geider & La Roche, 2002). However, given the high DNA content of about 29.9 pg
DNA per cell in A. minutum (Figueroa et al., 2010) and the approximate elemental composition of DNA (Geider & La Roche, 2002), the genomic DNA alone should account for about 56% of the intracellular P in stationary phase P-limited cultures
(0.16+/-0.03 pmol cell-1).
Stationary phase in control cultures
Even with abundant aeration, high pH in batch cultures and natural populations of microalgae is both a cause and indicator of insufficient biologically available dissolved
C to sustain further growth (Berman-Frank et al., 1994; Lundholm et al., 2005).Growth in the stationary-phase control cultures, where N- and P-nutrients were replete, was apparently limited by availability of dissolved CO2, as indicated by the high pH in the culture vessels (Fig. 2) and the resumption of growth after transfer to the small-volume follow-on culture. Limitation by low dissolved inorganic carbon is a phenomenon often encountered in stationary-phase batch cultures, but it has also been reported from Manuscript 4 133 dinoflagellate blooms in freshwater (Berman-Frank et al., 1994) and brackish water
(Hansen, 2002). In addition to its effect on carbon availability, high pH in itself has been shown to be a possible limiting factor for dinoflagellate growth (Hansen et al.,
2007), an effect supposedly mediated by a partial inhibition of cellular proton pumps leading to increased intracellular pH which can disrupt pH-sensitive enzymatic processes and lead to cell leakage (Søderberg & Hansen, 2007). Carbon or pH limitation of dinoflagellate growth is unlikely to occur in marine ecosystems, due to higher seawater buffer capacity (see Wolf-Gladrow & Riebesell, 1997). However, A. minutum can occur and also bloom in brackish coastal waters (Hwang & Lu, 2000;
Piumsomboon et al., 2001) and therefore responses to high pH and low CO2 availability are potentially ecologically relevant.
Photosynthetic performance
The potential quantum efficiency of Photosystem (PS)II, measured as Fv/Fm , is a sensitive indicator of photosynthetic performance and as such is often used as a general stress indicator for photosynthetic cells (Nedbal et al., 2000; Kim et al., 2006; Hsu,
2007; Tillmann et al., 2008). The Fv/Fm increase in exponentially growing control- and
P-limited cultures was associated with increasing cell concentrations (Fig. 3), and apparently results from acclimation to decreasing light availability in increasingly dense cultures. Similar effects are known from the green macroalga Cladophora sp., where increased Fv/Fm values are reported for light-limited environmental samples (Hiriart-
Baer et al., 2008), and from cultured isolates (clades A, B and F) of the symbiotic dinoflagellate Symbiodinium, in which acclimatisation to high light was associated with reduced Fv/Fm values (Robison & Warner, 2006).
The decrease in Fv/Fm measured in all stationary cultures may correspond to a higher proportion of damaged PSII centres due to impaired repair mechanisms (Lippemeier et Manuscript 4 134 al., 2001; Takahashi & Murata, 2008) or to down-regulation of photosynthesis- associated processes in non-growing cells. The lower demand for carbon compounds,
ATP and redox equivalents (see Geider et al., 1993) in combination with the reduced availability of dissolved CO2 at higher pH values in older cultures, can lead to a diversion of photosynthetic electrons to oxygen, resulting in the production of reactive oxygen species (ROS) (Vardi et al., 1999). Limitation of photosynthetic CO2 fixation can decreases the consumption of the reducing agent NADPH, potentially leading to depletion of its reduced form NADP+, the major acceptor of electrons for Photosystem
I. This increases the transfer of electrons from PSI to molecular oxygen. The generated
ROS impede the repair of photodamaged PSII by inhibiting the synthesis of new D1 protein at the elongation step of translation, leading to photoinhibition (reviewed by
Takahashi & Murata, 2008).
Factors influencing toxin content
Similar to other studies on various PSP toxin-producing Alexandrium species (John &
Flynn, 2000; Lippemeier et al., 2003; Leong et al., 2004), intracellular PSP toxin content in our cultures was closely linked to nutritional status. This sensitivity to nutrient limitation has been attributed to variation in intracellular concentrations of arginine (John & Flynn, 2000), which is a biosynthetic precursor of PSP toxin biosynthesis in cyanobacteria and likely also in dinoflagellates (Shimizu, 1982;
Kellmann & Neilan, 2007). Anderson et al. (1990) found that under most growth conditions, in laboratory cultures of A. fundyense, cellular concentrations of free arginine were low when toxin content peaked but increased rapidly as toxin content declined. This relationship also held for P-limited cultures. While intracellular toxin quota cannot be interpreted as a direct measure for the rate of toxin production
(Cembella, 1998), P-limitation in Alexandrium tends to lead to an increase in toxin Manuscript 4 135 productivity not only on a cellular but also on a culture volume basis (Lippemeier et al.,
2003). Anderson et al. (1990) explained this observation by the existence of a saxitoxin biosynthetic pathway that continues to operate even after the cessation of cell division, and which then depletes cellular arginine pools with greatly reduced competition from other pathways. Lippemeier et al. (2003) attributed the elevated toxin synthesis during P limitation to a potential arrest of cell cycle in G1, which is the cell cycle stage in which toxin production occurs (Taroncher-Oldenburg et al., 1997). This arrest in G1 would imply a continuous expression of G1-specific genes, which should include at least some of the genes coding for PSP toxin biosynthetic enzymes.
Allelochemical activity
Allelochemical activity against Rhodomonas salina, unlike PSP toxin content, did not respond in a notably treatment-specific pattern (Fig. 4b). In contrast to the situation in the haptophyte Prymnesium parvum, in which lytic activity is considerably induced by both N- and P-starvation (Granéli & Johansson, 2003), the pattern observed in A. minutum is more reminiscent of an accumulation with culture age that might be secondarily influenced by nutrient availability (Fig. 5b). An induction by growth limitation acting non-discriminately under P-, N- and pH- or C-limitation cannot be ruled out. In order to further illuminate the factors influencing synthesis and accumulation of the lytic compounds, highly targeted datasets combining a high number of measurements of allelochemical activity with finely graded variations of the physiological parameters seem most promising. The elucidation of the chemical composition and structure of those compounds is currently under way (Ma et al., 2009).
Gene expression in A. minutum
Dinoflagellates are evolutionarily distant from most model organisms, even among apicomplexa. Functional annotation of their genes is therefore often difficult (John et Manuscript 4 136 al., 2004). With the A. minutum EST library herein analysed, BLAST-based automated annotation as a starting point for manual annotation was available for only 28% of the sequences (Manuscript 1). In any case, physiological responses of the toxigenic dinoflagellate A. minutum, including growth kinetics and nutrient-dependent limitation,
PSP toxin biosynthesis and cell content, and allelochemical activity, are reflected in comparisons of gene expression among cultures grown under different environmental regimes. While this restricted availability of genomic information precluded pathway reconstruction, we nevertheless observed patterns characteristic for the processes involved in acclimation to the different treatments.
Growth stage dependent gene expression across all treatments
The high number of genes consistently up- or down-regulated in all stationary-phase cultures when compared to the exponentially growing control (Table 2b) mirrors the fundamental physiological differences between the different culture growth stages.
Gene expression differed considerably more among growth stages than between different treatments at the same growth stage (Tables 2a, 2b, 3), demonstrating an essential dissimilarity between actively growing and non-growing cells. An equivalent effect is documented in yeast, where the stages of fermentation, which correspond to the different growth stages in batch cultures, can affect the overall gene expression to a considerably greater degree than the fermentation medium or even the strain examined
(Rossouw & Bauer, 2009).
Not unexpectedly, the largest functional group among the genes higher expressed in exponential phase were genes involved in translational processes (Table 4). This is in agreement with the high number of sequences involved in protein synthesis and translational regulation identified in a recent study in A. catenella, which involved a gene expression library of exponential-phase A. catenella subtracted with a stationary- Manuscript 4 137 phase sample (Toulza et al., 2010). These authors considered the expression of the ribosomal component 5.8S rRNA as reflecting the whole-cell metabolic activity.
Following this suggestion, the reduced expression of 14 ribosome-connected sequences is an indication of reduced translational and metabolic activity associated with stationary phase. While a high incidence of post-transcriptional regulation in dinoflagellates has frequently been reported (Rossini et al., 2003; Lidie, 2007; Lapointe
& Morse, 2008), we found only one translation elongation factor-like protein and two splicing-related proteins to be down-regulated across all treatments.
In all stationary-phase treatments, gene expression patterns revealed evidence of down- regulation of photosystem components. Reduction of the number of photosystem reaction centres, otherwise known as a photoacclimation strategy (e.g. Ragni et al.,
2010), is apparently linked to the reduced Fv/Fm values in stationary-phase cultures.
This is likely to be either a response to photoinhibition due to ROS production linked to reduced photosynthetic CO2 fixation, or an acclimatisation response to avoid such damage. The main causes for the stationary-phase reduction in carbon fixation differ among treatments. In control and N-limited cultures, this effect may be directly caused by limiting CO2 concentrations (see above) and reduced amounts of the CO2-fixing enzyme (Huang et al., 2004), respectively. The reason for the reduced carbon fixation in
P-limited cultures is less well understood, but appears to be related to a decrease in photosynthetic electron transport capacity, which decreases the ATP content and hence
CO2 fixation, while increasing the likelihood of damage from excess absorbed excitation energy (Moseley et al., 2006; Lin et al., 2009b). Both photosynthetic performance and the related gene expression are most affected in the N-limitation treatment. Manuscript 4 138
The most prevalent functional group among the genes higher expressed in all of the stationary-phase cultures were sequences involved in intracellular signalling, which might be due to an increase in stress-related effects. Like the differential expression of translation – and photosynthesis-related gene expression, the importance of intracellular signalling-related sequences has also been noted in A. catenella (Toulza et al., 2010).
As the group of growth-state indicative genes identified here is correlated with the difference between exponentially growing cells and cells limited by different environmental factors, it provides an interesting starting point to identify marker genes or marker gene expression profiles to identify the level of growth limitation in mesocosm experiments or in potential A. minutum bloom populations.
Gene expression linked to stationary phase in control cultures
While a high number of genes were differentially expressed between exponentially growing controls and the same cultures in stationary phase, significant expression differences between the stationary-phase samples from different treatments were rare
(Table 2b). Of the 8 genes identified as specifically down-regulated in the stationary- phase control cultures as compared to both the exponential-phase control and the nutrient-limited cultures, three sequences were identified as ribosomal proteins, indicating a further down-regulation of the translational machinery when compared to the nutrient-limited cultures (Table 5).
Growth in these cultures was apparently limited by availability of dissolved organic carbon (see above). The typical dinoflagellate response to a reduced concentration of dissolved CO2 is an increase in amount and per-cell-activity of the enzymes involved in the dehydration of HCO3, the extracellular and intracellular carbonic anhydrases (CAs)
(Berman-Frank et al., 1994; Ratti et al., 2007; Lapointe et al., 2008). We identified an
H[WUDFHOOXODU į-CA similar to that of the bloom-forming marine dinoflagellate Manuscript 4 139
Lingulodinium polyedrum (Lapointe et al., 2008; see Toulza et al., 2010), as well as an intracellular CA, which were both significantly higher expressed in the stationary-phase control culture than in the exponentially growing control (supplementary Table 2).
Nutrient limitation-related gene expression patterns
Metabolic pathways and related gene expression conceivably affected by nutrient supply include mechanisms of active transport, nutrient assimilation and sequestration, as well as biosynthetic pathways leading to core components (proteins, nucleic acids, lipids) and secondary metabolites. Indeed, this is reflected in the gene expression data for A. minutum, as 23% of the genes tested were differentially expressed between at least one of the nutrient-limiting treatments and exponentially growing control cultures.
Most of these changes were associated with the difference between exponentially growing and stationary-phase cultures irrespective of the limiting factor, but 87 genes specifically responding to N- or P-starvation were identified (Tables 3, 6).
The most conspicuous group among these nutrient-limitation responsive sequences were the chloroplast-associated genes differentially regulated under N-limitation. For example, 8 mRNAs encoding cytochromes and photosystem proteins were on average
9.3 times less expressed under N-limitation than in the exponentially growing control, and those for two chlorophyll a-c-binding proteins and a ribulose-bisphosphate carboxylase (RubBisCO) were up-regulated by an average factor of 5.4 (Table 6). This is consistent with the marked N-dependency of these two core components of the photosynthetic apparatus. The key carbon fixation enzyme RubBisCO catalyzes the rate-limiting step of light-independent photosynthetic reaction and is generally the most abundant protein in photosynthetic organisms. Levels of RubBisCO can be severely compromised under N-limitation (Sims et al., 1998; Huang et al., 2004), which can limit overall photosynthesis (Parry et al., 2008). The reduced CO2 fixation can lead to Manuscript 4 140 substantially increased ROS production (see above) and cause enhanced photoinhibion
(Takahashi & Murata, 2008). An up-regulation of RubBisCO on the mRNA level might partially compensate this effect. Synthesis of the chlorophyll tetrapyrrole skeleton is dependent on the availability of amino acid precursors (reviewed in Nogaj et al., 2005), and lower chlorophyll quotas in N-limited cells are common (Verhoeven et al., 1997;
Sciandra et al., 2000; de Groot et al., 2003). This can be associated with reduced amounts of light harvesting complexes, while other components of the light harvesting machinery shift abundance (Peltier & Schmidt, 1991). The observed adjustments of the mRNA levels of chlorophyll-binding proteins in connection with reduced mRNA amounts of other photosystem proteins and different cytochromes is consistent with these phenomena. They are reflected in the decreased potential quantum efficiency of
Photosystem 2 (PSII) (Fig. 4) in the nitrogen-limited cultures. Similar strong effects of
N-starvation on photosynthetic performance are known from a variety of phytoplankton species (Flynn et al., 1994; Lippemeier et al., 2001; Juhl, 2005).
Transition to stationary phase
The 5 genes up-regulated at the transition to stationary phase in all treatments (Table 3) are associated with the onset of limitation or with a slowing of growth. As such, they constitute putative candidates as markers for the onset of growth limitation and potentially bloom breakdown.
In Alexandrium tamarense, nutrient limitation can lead to bloom breakdown by induction of resting cyst formation, which tends to remove considerable fractions of the vegetative population (Anderson & Lindquist, 1985; Ichimi et al., 2001; Yamamoto &
Seike, 2003). While N- and P-limitation can promote cyst production in A. minutum under laboratory conditions (Blanco, 1995; Figueroa et al., 2007), A. minutum bloom decline cannot usually be attributed to encystment (Garcés et al., 2004), as cyst Manuscript 4 141 production in bloom populations apparently is a constant process, involving a continual small percentage of cells throughout most of the bloom duration (Garcés et al., 2004;
Pitcher et al., 2007).
Nevertheless, in addition to the obvious implications of a marker for restricted growth, physiological state might influence the susceptibility of A. minutum to parasites and pathogens (Llaveria et al., 2010). This would indicate an increased likelihood of bloom termination by otherwise tolerated (but see Chambouvet et al., 2008) levels of infection
(Figueroa et al., 2008). Furthermore, a decrease in growth rate can lead to significant reductions of the dinoflagellate population by microzooplankton grazing (Calbet et al.,
2003), which might otherwise have little effect on an established bloom (Van Lenning et al., 2007; Estrada et al., 2010).
The genes associated with the transition to stationary phase included a serine/threonine- protein phosphatase, potentially a regulator of enzyme activity, and an ABC transporter probably involved in active transport across membranes. The transporter sequence was the only one of the transition phase-associated sequences that was not identified as associated with stationary phase as well. This reinforces the potential utility of these genes as markers for early stages of limitation.
Toxin-related gene expression
Our gene expression results contribute little to the elucidation of the regulation of PSP toxin content. While more than half of the genes differentially expressed between the highly toxin-containing P-limited cultures and the low-toxin N-limited cultures could be annotated, ,all inferred functions suggested involvement in the regulation of core metabolic responses (Table 7). Nevertheless, the differential expression pattern of two of the non-annotatable sequences was consistent with an involvement in the regulation of PSP toxin levels: Amin_21a03r was up-regulated under both beginning and severe P- Manuscript 4 142 limitation and down-regulated under severe N-limitation when compared to any other condition, and Amin_78e07r was higher expressed under P-limited than in the N- limited conditions, less expressed under N-limited than under stationary phase control conditions, and not significantly differentially expressed any other comparison (Table
7). Additionally, earlier BLAST comparisons had identified a sequence similar to
Amin_78e07r in the PSP toxin-producing related species A. tamarense, but not in any other dinoflagellate, including several other PSP toxin producing and non-PSP-toxin- producing Alexandrium species (Manuscript 1). However, as these BLAST comparisons were based on non-exhaustive EST databases, and absence of a sequence in these databases is not conclusive evidence of absence in the respective organism.
Nevertheless, as these two sequences are genes of unknown function with an expression pattern suggestive of involvement in PSP toxin regulation, they warrant further examination in more targeted experiments.
Gene expression – general trends
The data presented here show substantial changes in the A. minutum transcriptome associated with medium-term differences in growth and nutrient status, adding to a complicated pattern of transcriptomic and translation-level regulation of gene expression in dinoflagellates:
We identified 41% of all tested sequences to be differentially expressed between any of the growth-limited treatments and the exponential-phase control samples (Table 2 a,b).
This is a somewhat higher change rate than reported from the related species A. tamarense in a recent MPSS study (Moustafa et al., 2010): In A. tamarense, 73% of transcripts remained uniformly abundant irrespective of N-or P-limitation or the presence or absence of bacteria. Whether this difference is due to true differences in the Manuscript 4 143 prevalence of transcriptome-level gene regulation, or due to the different experimental setups, remains to be tested.
In different dinoflagellate species, regulation of the mRNA pool seems to be mainly found in association with longer-term physiological differences, such as adaptation to different light regimes on a timescale of days to weeks (Roman et al., 1988; ten Lohuis
& Miller, 1998),or during different stages of the sexual life-cycle (Hosoi-Tanabe et al.,
2005). While part of the gene regulation associated with different stages of the cell cycle is associated with differences in transcript abundance (Taroncher-Oldenburg &
Anderson, 2000), several studies confirmed translational regulation of marker genes over the circadian cycle (Mittag et al., 1994; Rossini et al., 2003; Lapointe & Morse,
2008). A microarray investigation revealed that about 10% of the K. brevis transcriptome changes over the duration of the light-dark cycle. Genes involved in posttranscriptional processing of RNA and protein turnover were unusually highly represented among these sequences. On the other hand, cell cycle genes did not seem to be transcriptionally regulated (Van Dolah et al., 2007). In the same species, acute peroxide stress lead to conspicuous changes in protein levels which in most cases were not associated with corresponding changes in transcript levels (Lidie, 2007). Instead, short-term shock response, which was monitored during the first 240 min after shock treatment, seemed to be associated with changing representations of stress-related mRNAs in the actively translated pool. In a similar stress study on a slightly longer timescale, about 4% of P. lunula genes were differentially expressed on the transcriptome level 6h after beginning redox-stress treatments (Okamoto & Hastings,
2003). The transcriptomic response affected several groups of conventional stress- response genes, including several kinases, proteins involved in protein ubiquination and degradation, and transcription factors. Manuscript 4 144
These differences might be strongly dependent on the timescale investigated: a strong translational component of the short-time response to shock treatments is also known from other organisms such as yeast (Melamed et al., 2008), in which the inhibition of protein synthesis caused by salt shock is reversed over a timescale of 5h. In human cell cultures under hypoxia, rapid translational responses are mediated by the translation factor elF2, resulting in a drop in the average number of ribosomes per translated transcript. This effect reaches a maximum at 1–2 h. The response to prolonged anoxia
(~16 h) is associated with disruption of mRNA cap-binding complex, resulting in a reduction in fraction of mRNA found within polysomes. While the overall result is an inhibition of global mRNA translation, specific mRNAs are preferentially translated or even translationally induced (Koritzinsky et al., 2006). Another factor differentially influencing the translation of mRNAs is the activity of microRNAs, single-stranded
RNA molecules that form part of a ribonucleoprotein complex which selectively mediates the degradation, destabilisation or translational inhibition of target mRNAs
(reviewed in Winter et al., 2009). MicroRNAs are well studied in animals and plants, but have also been identified in various protists (Lin et al., 2009a, and references therein).
In general, translational gene expression is a phenomenon well known from model organisms and is probably especially prominent during rapid stress response.
Nevertheless, available data suggest a particularly strong component of post- transcriptional regulation in dinoflagellate gene expression, not only during shock response but also associated with other short-term physiological changes such as differences over the light-dark cycle. Even in long-term adaptation genes involved in the regulation of translation and protein degradation tend to be among the most highly regulated functional groups, but this is not associated with a systematic lack of regulation on the transcriptomic level. Manuscript 4 145
In analogy to the situation in trypanosomes, the phenomenon of mRNA transsplicing in dinoflagellates has been suggested to be associated with post-transcriptional control of gene expression (Bachvaroff & Place, 2008; Monroe & Van Dolah, 2008). Our data on the percentages of differentially expressed A. mintum sequences which contain the typical transsplicing-associated spliced leader sequence suggests that spliced-leader transsplicing in dinoflagellates is not associated with a lack of regulation at the transcriptional level. Whereas 4.7% of all the sequences in our normalised EST library contained a spliced leader sequence, the same was true for 4.4% of the genes differentially expressed between one of the treatments and the exponential-phase control samples, and for 5.8% of those showing the same trend of differential expression in several comparisons. As many of these ESTs are incomplete and 3’ end-biased representations of the corresponding mRNAs, SL sequences are very probably hugely underrepresented. The mentioned percentages should therefore not be taken as indicative of the prevalence of SLs in A. minutum mRNAs. While the equal representation of spliced leader sequences among the differentially expressed genes does not imply any specific function of this sequence, the data presented here are in agreement with those of Zhang and Lin (2009), who found spliced-leaders associated with all sequences examined. This indicates that even if SL trans-splicing is involved in the post-transcriptional regulation of gene expression, e.g. in mRNA stabilisation or in the recruitment into the actively translated mRNA pool, it is probably not restricted to constitutively transcribed genes.
Conclusions
The dataset presented here constitutes a comprehensive series of measurements related to bloom-relevant factors such as toxin content, allelochemical activity, nutrient limitation and growth. While the growth stages of batch cultures are a rather crude Manuscript 4 146 model for the stages of bloom development, they nevertheless permit a comparison between actively proliferating and non-growing cells. Using extensive cross- comparisons of the differential gene expression responses, we identified narrow gene expression patterns linked with specific physiological factors such as exponential growth, cessation of growth or nutrient limitation.
Acknowledgements
Thanks to Christiane Lorenzen for help with the nutrient measurements and to Annegret
Müller and Bernd Krock for help with the PSP toxin measurements. This study was financed by EU-project ESTTAL (GOCE-CT2004-511154) and Marine Genomics
(GOCE-CT-2004-505403) and the PACES research program of the Alfred Wegener
Institute as part of the Helmholtz Foundation initiative in Earth and Environment. Manuscript 4 147
References
.
ANDERSON, C. R., SIEGEL, D. A., BRZEZINSKI, M. A., GUILLOCHEAU, N. (2008). Controls on temporal patterns in phytoplankton community structure in the Santa Barbara Channel, California. Journal of Geophysical Research-Oceans 113(C4).
ANDERSON, D. M., LINDQUIST, N. L. (1985). Time-course measurements of phosphorus depletion and cyst formation in the dinoflagellate Gonyaulax tamarensis Lebour. Journal of Experimental Marine Biology and Ecology 86(1): 1-13.
ANDERSON, D. M., KULIS, D. M., SULLIVAN, J. J., HALL, S., LEE, C. (1990). Dynamics and physiology of saxitoxin production by the dinoflagellates Alexandrium spp. Marine Biology 104: 511- 524.
ANDERSON, D. M., GRABHER, A., HERZOG., M. (1992). Separation of coding sequences from structural DNA in the dinoflagellate Crypthecodinium cohnii. Molecular Marine Biology and Biotechnology 1: 89-96.
BACHVAROFF, T., PLACE, A. (2008). From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae. PLoS ONE 3(8).
BERMAN-FRANK, I., ZOHARY, T., EREZ, J., DUBINSKY, Z. (1994). CO2 availability, carbonic anhydrase, and the annual dinoflagellate bloom in Lake Kinneret. Limnology and Oceanography 39(8): 1822-1834.
BLANCO, J. (1995). Cyst production in four species of neritic dinoflagellates. Journal Of Plankton Research 17(1): 165-182.
BRAVO, I., VILA, M., MASÓ, M., FIGUEROA, R. I., RAMILO, I. (2008). Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches. Harmful Algae 7(4): 515.
CALBET, A., VAQUÉ, D., FELIPE, J., VILA, M., SALA, M. M., ALCARAZ, M., ESTRADA, M. (2003). Relative grazing impact of microzooplankton and mesozooplankton on a bloom of the toxic dinoflagellate Alexandrium minutum. Marine Ecology-Progress Series 259: 303-309.
CEMBELLA, A. D. (1998). Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. In Physiological Ecology of Harmful Algal Blooms (D. M. ANDERSON, A. D. CEMBELLA, G. M. HALLEGRAEFF editors), pp. 381-403. Springer Verlag, Berlin, Heidelberg, New York.
CEMBELLA, A. D. (2003). Chemical ecology of eukaryotic microalgae in marine ecosystems. Phycologia 42(4): 420-447.
CEMBELLA, A. D., JOHN, U. (2006). Molecular physiology of toxin production and growth regulation in harmful algae. In Ecology of Harmful Algae (E. GRANÉLI , J. T. TURNER editors), pp. 215-227. Springer Berlin Heidelberg.
CHAMBOUVET, A., MORIN, P., MARIE, D., GUILLOU, L. (2008). Control of toxic marine dinoflagellate blooms by serial parasitic killers. Science 322(5905): 1254-1257.
CHANG, F. H., ANDERSON, D. M., KULIS, D. M., TILL, D. G. (1997). Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand. Toxicon 35: 393-409.
CHANG, F. H., MCCLEAN, M. (1997). Growth responses of Alexandrium minutum (Dinophyceae) as a function of three different nitrogen sources and irradiance. New Zealand Journal of Marine and Freshwater Research 31: 1-7. Manuscript 4 148
DE GROOT, C. C., VAN DEN BOOGAARD, R., MARCELIS, L. F. M., HARBINSON, J., LAMBERS, H. (2003). Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation. Journal of Experimental Botany 54(389): 1957-1967.
ESCHBACH, E., JOHN, U., RECKERMANN, M., CEMBELLA, A. D., EDVARDSEN, B., MEDLIN, L. K. (2005). Cell cycle dependent expression of toxicity by the ichthyotoxic prymnesiophyte Chrysochromulina polylepis. Aquatic Microbial Ecology 39: 85–95.
ESTRADA, M., SOLÉ, J., ANGLÈS, S., GARCÉS, E. (2010). The role of resting cysts in Alexandrium minutum population dynamics. Deep-sea research. Part II, Topical studies in oceanography 57(3-4): 308- 321.
FIGUEROA, R. I., GARCÉS, E., BRAVO, I. (2007). Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales, Dinophyceae) in culture. Journal of Phycology 43(5): 1039-1053.
FIGUEROA, R. I., GARCÉS, E., MASSANA, R., CAMP, J. (2008). Description, host-specificity, and strain selectivity of the dinoflagellate parasite Parvilucifera sinerae sp nov (Perkinsozoa). Protist 159(4): 563-578.
FIGUEROA, R. I., GARCÉS, E., BRAVO, I. (2010). The use of flow cytometry for species identification and life-cycle studies in dinoflagellates. Deep-sea research. Part II, Topical studies in oceanography 57(3- 4): 301-307.
FLYNN, K., FRANCO, J. M., FERNÁNDEZ, P., REGUERA, B., ZAPATA, M., WOOD, G., FLYNN, K. J. (1994). Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress. Marine Ecology Progress Series 111: 99-109.
FLYNN, K., JONES, K. J., FLYNN, K. J. (1996). Comparisons among species of Alexandrium (Dinophyceae) grown in nitrogen- or phosphorus-limiting batch culture. Marine Biology 126(1): 9-18.
GARCÉS, E., BRAVO, I., VILA, M., FIGUEROA, R. I., MASÓ, M., SAMPEDRO, N. (2004). Relationship between vegetative cells and cyst production during Alexandrium minutum bloom in Arenys de Mar harbour (NW Mediterranean). Journal Of Plankton Research 26(6): 637-645.
GEIDER, R. J., ROCHE, J., GREENE, R. M., OLAIZOLA, M. (1993). Response of the photosynthetic apparatus of Phaeodactylum tricornutum (Bacillariophyceae) to nitrate, phosphate, or iron starvation. Journal of Phycology 29(6): 755-766.
GEIDER, R. J., LA ROCHE, J. (2002). Redfield revisited: variability of C : N : P in marine microalgae and its biochemical basis. European Journal of Phycology 37(1): 1-17.
GRANÉLI, E., JOHANSSON, N. (2003). Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions. Harmful Algae 2(2): 135-145.
HANSEN, P. J. (2002). Effect of high pH on the growth and survival of marine phytoplankton: implications for species succession. Aquatic Microbial Ecology 28(3): 279-288.
HANSEN, P. J., LUNDHOLM, N., ROST, B. (2007). Growth limitation in marine red-tide dinoflagellates: effects of pH versus inorganic carbon availability. Marine Ecology Progress Series 334: 63-71.
HIRIART-BAER, V. P., ARCISZEWSKI, T. J., MALKIN, S. Y., GUILDFORD, S. J., HECKY, R. E. (2008). Use of pulse-amplitude-modulated fluorescence to assess the physiological status of Cladophora sp along a water quality gradient. Journal of Phycology 44(6): 1604-1613.
HOBBIE, J. E., DALEY, R. J., JASPER, S. (1977). Use of nuclepore filters for counting bacteria by fluorescence microscopy. Applied and Environmental Microbiology 33: 1225-1228. Manuscript 4 149
HOSOI-TANABE, S., TOMISHIMA, S., NAGAI, S., SAKO, Y. (2005). Identification of a gene induced in conjugation-promoted cells of toxic marine dinoflagellates Alexandrium tamarense and Alexandrium catenella using differential display analysis. FEMS Microbiology Letters 251(1): 161-168.
HSU, B. D. (2007). On the possibility of using a chlorophyll fluorescence parameter as an indirect indicator for the growth of Phalaenopsis seedlings. Plant Science 172(3): 604-608.
HUANG, Z. A., JIANG, D. A., YANG, Y., SUN, J. W., JIN, S. H. (2004). Effects of nitrogen deficiency on gas exchange, chlorophyll fluorescence, and antioxidant enzymes in leaves of rice plants. Photosynthetica 42(3): 357-364.
HWANG, D. F., LU, Y. H. (2000). Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum. Toxicon 38(11): 1491-1503.
ICHIMI, K., YAMASAKI, M., OKUMURA, Y., SUZUKI, T. (2001). The growth and cyst formation of a toxic dinoflagellate, Alexandrium tamarense, at low water temperatures in northeastern Japan. Journal of Experimental Marine Biology and Ecology 261(1): 17-29.
JOHN, E. H., FLYNN, K. J. (2000). Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N:P supply ratios on internal toxin and nutrient levels. European Journal of Phycology 35(1): 11.
JOHN, U., MOCK, T., VALENTIN, K., CEMBELLA, A. D., MEDLIN, L. K. (2004). Dinoflagellates come from outer space but haptophytes and diatoms do not. In Proceedings of Harmful Algae 2002, St. Pete Beach, Florida, USA. 21–25.10.2002 (K. A. STEIDINGER, J. H. LANDSBERG, C. R. TOMAS, G. A. VARGO editors)
JUHL, A. R. (2005). Growth rates and elemental composition of Alexandrium monilatum, a red-tide dinoflagellate. Harmful Algae 4(2): 287-295.
KATTNER, G., BROCKMANN, U. H. (1980). Semi-automated methods for the determination of particulate phosphorus in the marine environment. Fresenius' Zeitschrift für analytische Chemie 301: 14- 16.
KELLER, M. D., SELVIN, R. C., CLAUS, W., GUILLARD, R. R. L. (1987). Media for the culture of oceanic ultraphytoplankton. Journal of Phycology 23(4): 633-638.
KELLMANN, R., NEILAN, B. A. (2007). Biochemical characterization of paralytic shellfish toxin biosynthesis in vitro. Journal of Phycology 43(3): 497-508.
KIM, K. S., GIACOMELII, G. A., SASE, S., SON, J. E., NAM, S. W., NAKAZAWA, F. (2006). Optimization of growth environment in a plant production facility using a chlorophyll fluorescence method. Jarq-Japan Agricultural Research Quarterly 40(2): 149-156.
KORITZINSKY, M., MAGAGNIN, M. G., VAN DEN BEUCKEN, T., SEIGNEURIC, R., SAVELKOULS, K., DOSTIE, J., PYRONNET, S., KAUFMAN, R. J., WEPPLER, S. A., VONCKEN, J. W., LAMBIN, P., KOUMENIS, C., SONENBERG, N., WOUTERS, B. G. (2006). Gene expression during acute and prolonged hypoxia is regulated by distinct mechanisms of translational control. Embo Journal 25(5): 1114-1125.
KROCK, B., SEGUEL, C. G., CEMBELLA, A. D. (2007). Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry. Harmful Algae 6(5): 734-744.
LABRY, C., ERARD-LE DENN, E., CHAPELLE, A., FAUCHOT, J., YOUENOU, A., CRASSOUS, M. P., LE GRAND, J., LORGEOUX, B. (2008). Competition for phosphorus between two dinoflagellates: A toxic Alexandrium minutum and a non-toxic Heterocapsa triquetra. Journal of Experimental Marine Biology and Ecology 358(2): 124-135.
LAJEUNESSE, T. C., LAMBERT, G., ANDERSEN, R. A., COFFROTH, M. A., GALBRAITH, D. W. (2005). Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. Journal of Phycology 41(4): 880-886. Manuscript 4 150
/$32,17( 0 0$&.(1=,(7 ' % 0256( ' $Q H[WHUQDO į-carbonic anhydrase in a free-living marine dinoflagellate may circumvent diffusion-limited carbon acquisition. Plant Physiology 147(3): 1427-1436.
LAPOINTE, M., MORSE, D. (2008). Reassessing the role of a 3 '-UTR-binding translational inhibitor in regulation of circadian bioluminescence rhythm in the dinoflagellate Gonyaulax. Biological Chemistry 389(1): 13-19.
LEONG, S. C. Y., MURATA, A., NAGASHIMA, Y., TAGUCHI, S. (2004). Variability in toxicity of the dinoflagellate Alexandrium tamarense in response to different nitrogen sources and concentrations. Toxicon 43(4): 407.
LIDIE, K. B. (2007). Characterization and regulation of gene expression networks in response to acute environmental stress in the Florida Red Tide Dinoflagellate, Karenia brevis. In Molecular and Cellular Biology and Pathobiology Department, p. 250. Medical University of South Carolina.
LIDIE, K. B., VAN DOLAH, F. M. (2007). Spliced leader RNA-mediated trans-splicing in a dinoflagellate, Karenia brevis. Journal Of Eukaryotic Microbiology 54(5): 427-435.
LIN, W. C., LI, S. C., LIN, W. C., SHIN, J. W., HU, S. N., YU, X. M., HUANG, T. Y., CHEN, S. C., CHEN, H. C., CHEN, S. J., HUANG, P. J., GAN, R. R. C., CHIU, C. H., TANG, P. (2009a). Identification of microRNA in the protist Trichomonas vaginalis. Genomics 93(5): 487-493.
LIN, Z. H., CHEN, L. S., CHEN, R. B., ZHANG, F. Z., JIANG, H. X., TANG, N. (2009b). CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply. BMC Plant Biology 9.
LIPPEMEIER, S., KLAUS, R. H., VANSELOW, K. H., HARTIG, P., COLIJN, F. (2001). In-line recording of PAM fluorescence of phytoplankton cultures as a new tool for studying effects of fluctuating nutrient supply on photosynthesis. European Journal of Phycology 36: 89-100.
LIPPEMEIER, S., FRAMPTON, D. M. F., BLACKBURN, S. I., GEIER, S. C., NEGRI, A. P. (2003). Influence of phosphorus limitation on toxicity and photosynthesis of Alexandrium minutum (Dinophyceae) monitored by in-line detection of variable chlorophyll fluorescence. Journal of Phycology 39(2): 320-331.
LIVOLANT, F., BOULIGAND, Y. (1978). New observations on the twisted arrangement of Dinoflagellate chromosomes. Chromosoma 68(1): 21.
LLAVERIA, G., GARCÉS, E., ROSS, O. N., FIGUEROA, R. I., SAMPEDRO, N., BERDALET, E. (2010). Small-scale turbulence can reduce parasite infectivity to dinoflagellates. Marine Ecology Progress Series 412: 45-56.
LUNDHOLM, N., HANSEN, P. J., KOTAKI, Y. (2005). Lack of allelopathic effects of the domoic acid- producing marine diatom Pseudo-nitzschia multiseries. Marine Ecology-Progress Series 288: 21-33.
MA, H. Y., KROCK, B., TILLMANN, U., CEMBELLA, A. (2009). Preliminary characterization of extracellular allelochemicals of the toxic marine dinoflagellate Alexandrium tamarense using a Rhodomonas salina bioassay. Marine Drugs 7(4): 497-522.
MAGUER, J. F., L'HELGUEN, S., MADEC, C., LABRY, C., LE CORRE, P. (2007). Nitrogen uptake and assimilation kinetics in Alexandrium minutum (Dinophyceae): Effect of N-limited growth rate on nitrate and ammonium interactions. Journal of Phycology 43(2): 295-303.
MCEWAN, M., HUMAYUN, R., SLAMOVITS, C. H., KEELING, P. J. (2008). Nuclear genome sequence survey of the dinoflagellate Heterocapsa triquetra. Journal Of Eukaryotic Microbiology 55(6): 530-535.
MELAMED, D., PNUELI, L., ARAVA, Y. (2008). Yeast translational response to high salinity: Global analysis reveals regulation at multiple levels. Rna-A Publication Of The Rna Society 14(7): 1337-1351. Manuscript 4 151
MITTAG, M., LEE, D. H., HASTINGS, J. W. (1994). Circadian expression of the luciferin-binding protein correlates with the binding of a protein to the 3' untranslated region of its mRNA. Proceedings of the National Academy of Sciences of the United States of America 91(12): 5257-5261.
MOCK, T., HOCH, N. (2005). Long-term temperature acclimation of photosynthesis in steady-state cultures of the polar diatom Fragilariopsis cylindrus. Photosynthesis Research 85(3): 307-317.
MONROE, E. A., VAN DOLAH, F. M. (2008). The toxic dinoflagellate Karenia brevis encodes novel type I-like polyketide synthases containing discrete catalytic domains. Protist 159(3): 471-482.
MORENO DÍAZ DE LA ESPINA, S., ALVERCA, E., CUADRADO, A., FRANCA, S. (2005). Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates. European Journal of Cell Biology 84(2-3): 137-149.
MOSELEY, J. L., CHANG, C. W., GROSSMAN, A. R. (2006). Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas reinhardtii. Eukaryotic Cell 5(1): 26-44.
MOUSTAFA, A., EVANS, A., KULIS, D., HACKETT , J., ERDNER, D., ANDERSON, D., BHATTACHARYA, D. (2010). Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence. PLoS ONE 5(3): e9688.
NEDBAL, L., SOUKUPOVA, J., WHITMARSH, J., TRTILEK, M. (2000). Postharvest imaging of chlorophyll fluorescence from lemons can be used to predict fruit quality. Photosynthetica 38(4): 571- 579.
NOGAJ, L. A., SRIVASTAVA, A., VAN LIS, R., BEALE, S. I. (2005). Cellular levels of glutamyl- tRNA reductase and glutamate-1-semialdehyde aminotransferase do not control chlorophyll synthesis in Chlamydomonas reinhardtii. Plant Physiology 139(1): 389-396.
OKAMOTO, O. K., HASTINGS, J. W. (2003). Genome-wide analysis of redox-regulated genes in a dinoflagellate. Gene 321: 73.
PARRY, M. A. J., KEYS, A. J., MADGWICK, P. J., CARMO-SILVA, A. E., ANDRALOJC, P. J. (2008). Rubisco regulation: a role for inhibitors. Journal of Experimental Botany 59(7): 1569-1580.
PELTIER, G., SCHMIDT, G. W. (1991). Chlororespiration: an adaptation to nitrogen deficiency in Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences of the United States of America 88(11): 4791-4795.
PITCHER, G. C., CEMBELLA, A. D., JOYCE, L. B., LARSEN, J., PROBYN, T. A., RUIZ SEBASTIÁN, C. (2007). The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition. Harmful Algae 6(6): 823- 836.
PIUMSOMBOON, A., SONGROOP, C., KUNGSUWAN, A., POLPUNTHIN, P. (2001). Species of the dinoflagellate genus Alexandrium (Gonyaulacales) in the gulf of Thailand. In Proceedings of the Ninth International Conference on Harmful Algal Blooms, Hobart, Australia. 7-11 February 2000 (G. M. HALLEGRAEFF, S. I. BLACKBURN, C. J. BOLCH, R. J. LEWIS editors)
RAGNI, M., AIRS, R., HENNIGE, S., SUGGETT, D., WARNER, M., GEIDER, R. (2010). PSII photoinhibition and photorepair in Symbiodinium (Pyrrhophyta) differs between thermally tolerant and sensitive phylotypes. Marine Ecology Progress Series 406: 57-70.
RATTI, S., GIORDANO, M., MORSE, D. (2007). CO2-concentrating mechanisms of the potentially toxic dinoflagellate Protoceratium reticulatum (Dinophyceae, Gonyaulacales). Journal of Phycology 43(4): 693-701.
REDFIELD, A. C. (1958). The biological control of chemical factors in the environment. American Scientist 46(3): 205-221. Manuscript 4 152
ROBISON, J. D., WARNER, M. E. (2006). Differential impacts of photoacclimation and thermal stress on the photobiology of four different phylotypes of Symbiodinium (Pyrrhophyta). Journal of Phycology 42(3): 568-579.
ROMAN, S. J., GOVIND, N. S., TRIPLETT, E. L., PRÉZELIN, B. B. (1988). Light regulation of peridinin-chlorophyll a-protein (PCP) complexes in the dinoflagellate, Glenodinium sp.: use of anti-PCP antibodies to detect PCP gene products in cells grown in different light conditions. Plant Physiology 88(3): 594-599.
ROSSINI, C., TAYLOR, W., FAGAN, T., HASTINGS, J. W. (2003). Lifetimes of mRNAs for clock- regulated proteins in a dinoflagellate. Chronobiology International 20(6): 963-976.
ROSSOUW, D., BAUER, F. F. (2009). Comparing the transcriptomes of wine yeast strains: toward understanding the interaction between environment and transcriptome during fermentation. Applied Microbiology and Biotechnology 84(5): 937-954.
SAEED, A. I., BHAGABATI, N. K., BRAISTED, J. C., LIANG, W., SHAROV, V., HOWE, E. A., LI, J., THIAGARAJAN, M., WHITE, J. A., QUACKENBUSH, J. (2006). TM4 microarray software suite. Methods in Enzymology Volume 411: 134-193.
SCIANDRA, A., LAZZARA, L., CLAUSTRE, H., BABIN, M. (2000). Responses of growth rate, pigment composition and optical properties of Cryptomonas sp to light and nitrogen stresses. Marine Ecology-Progress Series 201: 107-120.
SHIMIZU, Y. (1982). Recent progress in marine toxin research. Pure and Applied Chemistry.
SIMS, D. A., LUO, Y., SEEMANN, J. R. (1998). Comparison of photosynthetic acclimation to elevated CO2 and limited nitrogen supply in soybean. Plant, Cell and Environment 21(9): 945-952.
SLAMOVITS, C. H., KEELING, P. J. (2008). Widespread recycling of processed cDNAs in dinoflagellates. Current Biology 18(13): R550-R552.
SMAYDA, T. J. (1997). What is a bloom? A commentary. Limnology and Oceanography 42(5): 1132- 1136.
SØDERBERG, L. M., HANSEN, P. J. (2007). Growth limitation due to high pH and low inorganic carbon concentrations in temperate species of the dinoflagellate genus Ceratium. Marine Ecology- Progress Series 351: 103-112.
STOREY, J. D. (2003). The positive false discovery rate: A Bayesian interpretation and the q-value. Annals Of Statistics 31(6): 2013-2035.
TAKAHASHI, S., MURATA, N. (2008). How do environmental stresses accelerate photoinhibition? Trends In Plant Science 13(4): 178-182.
TARONCHER-OLDENBURG, G., KULIS, D. M., ANDERSON, D. M. (1997). Toxin variability during the cell cycle of the dinoflagellate Alexandrium fundyense. Limnology and Oceanography 42: 1178- 1188.
TARONCHER-OLDENBURG, G., ANDERSON, D. M. (2000). Identification and characterization of three differentially expressed genes, encoding S-adenosylhomocysteine hydrolase, methionine aminopeptidase, and a histone-like protein, in the toxic dinoflagellate Alexandrium fundyense. Applied and Environmental Microbiology 66(5): 2105-2112.
TEN LOHUIS, M. R., MILLER, D. J. (1998). Light-regulated transcription of genes encoding peridinin chlorophyll a proteins and the major intrinsic light-harvesting complex proteins in the dinoflagellate Amphidinium carterae Hulburt (Dinophycae) - Changes in cytosine methylation accompany photoadaptation. Plant Physiology 117(1): 189-196.
THOMPSON, P. A., BONHAM, P. I., SWADLING, K. M. (2008). Phytoplankton blooms in the Huon Estuary, Tasmania: top-down or bottom-up control? Journal Of Plankton Research 30(7): 735-753. Manuscript 4 153
TILLMANN, U., JOHN, U. (2002). Toxic effects of Alexandrium spp. on heterotrophic dinoflagellates: an allelochemical defence mechanism independent of PSP-toxin content. Marine Ecology Progress Series 230: 47-58.
TILLMANN, U., ALPERMANN, T., JOHN, U., CEMBELLA, A. (2008). Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists. Harmful Algae 7(1): 52-64.
TILLMANN, U., ELBRÄCHTER, M., KROCK, B., JOHN, U., CEMBELLA, A. (2009). Azadinium spinosum gen. et sp nov (Dinophyceae) identified as a primary producer of azaspiracid toxins. European Journal of Phycology 44(1): 63-79.
TOULZA, E., SHIN, M.-S., BLANC, G., AUDIC, S., LAABIR, M., COLLOS, Y., CLAVERIE, J.-M., GRZEBYK, D. (2010). Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella (Dinophyceae). Applied and Environmental Microbiology 76(13): 4521-4529.
TOUZET, N., FRANCO, J. M., RAINE, R. (2007). Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland. Toxicon 50(1): 106-119.
TUSHER, V. G., TIBSHIRANI, R., CHU, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proceedings of the National Academy of Sciences of the United States of America 98(9): 5116-5121.
VAN DOLAH, F. M., LIDIE, K. B., MOREY, J. S., BRUNELLE, S. A., RYAN, J. C., MONROE, E. A., HAYNES, B. L. (2007). Microarray analysis of diurnal- and circadian-regulated genes in the Florida red- tide dinoflagellate Karenia brevis (Dinophyceae). Journal of Phycology 43(4): 741-752.
VAN LENNING, K., VILA, M., MASÓ, M., GARCÉS, E., ANGLÈS, S., SAMPEDRO, N., MORALES- BLAKE, A., CAMP, J. (2007). Short-term variations in development of a recurrent toxic Alexandrium minutum - dominated dinoflagellate bloom induced by meteorological conditions. Journal of Phycology 43(5): 892-907.
VARDI, A., BERMAN-FRANK, I., ROZENBERG, T., HADAS, O., KAPLAN, A., LEVINE, A. (1999). Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO2 limitation and oxidative stress. Current Biology 9(18): 1061-1064.
VERHOEVEN, A. S., DEMMIGADAMS, B., ADAMS, W. W. (1997). Enhanced employment of the xanthophyll cycle and thermal energy dissipation in spinach exposed to high light and N stress. Plant Physiology 113(3): 817-824.
VILA, M., GIACOBBE, M. G., MASÓ, M., GANGEMI, E., PENNA, A., SAMPEDRO, N., AZZARO, F., CAMP, J., GALLUZZI, L. (2005). A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas. Harmful Algae 4(4): 673-695.
WINTER, J., JUNG, S., KELLER, S., GREGORY, R. I., DIEDERICHS, S. (2009). Many roads to maturity: microRNA biogenesis pathways and their regulation. Nature Cell Biology 11(3): 228-234.
WOLF-GLADROW, D., RIEBESELL, U. (1997). Diffusion and reactions in the vicinity of plankton: A refined model for inorganic carbon transport. Marine Chemistry 59(1-2): 17-34.
YALLOP, M. L. (2001). Distribution patterns and biomass estimates of diatoms and autotrophic dinoflagellates in the NE Atlantic during June and July 1996. Deep-sea research. Part II, Topical studies in oceanography 48(4-5): 825-844.
YAMAMOTO, T., TARUTANI, K. (1999). Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan. Phycological Research 47(1): 27-32.
YAMAMOTO, T., SEIKE, T. (2003). Modelling the population dynamics of the toxic dinoflagellate Alexandrium tamarense in Hiroshima Bay, Japan. II. Sensitivity to physical and biological parameters. Journal Of Plankton Research 25(1): 63-81. Manuscript 4 154
ZHANG, H., HOU, Y. B., MIRANDA, L., CAMPBELL, D. A., STURM, N. R., GAASTERLAND, T., LIN, S. J. (2007). Spliced leader RNA trans-splicing in dinoflagellates. Proceedings of the National Academy of Sciences of the United States of America 104(11): 4618-4623.
ZHANG, H., LIN, S. (2009). Retrieval of missing spliced leader in dinoflagellates. PLoS ONE 4(1). Manuscript 4 155
Figures
Figure 1 Growth kinetics of Alexandrium minutum AL3T in batch culture experiments: a) cell concentrations versus day after inoculation. Manuscript 4 156
b) growth rate (µ) versus day after inoculation (µ=ln(cell count (day2))-ln(cell count(day1)), lines: 2-day moving average. Manuscript 4 157
c) cell concentrations versus day after inoculation including nutrient-spiked (solid lines) and non-spiked (open symbols) run-on cultures. All values are mean ± standard deviation of biological triplicates. Manuscript 4 158
Figure 2 pH of the culture medium for various treatments given as mean ± standard deviation of biological triplicates. The last data point corresponds to the treatment- specific stationary-phase harvesting date. Manuscript 4 159
Figure 3 Nutrient status of cultures. a) Dissolved nutrients in culture medium. Manuscript 4 160
b) Intracellular nutrients. Manuscript 4 161
c) Element ratios. Average of biological triplicates, with standard deviation. Manuscript 4 162
Figure 4 Potential quantum efficiency of PSII, measured as dark-adapted Fv/Fm. Mean of triplicates, with standard deviation. Manuscript 4 163
Figure 5 Toxicity and allelochemical activity. a) Intracellular toxins (fmol cell-1) as measured by liquid chromatography with fluorescence detection (LC-FD). Mean of biological triplicates, with standard deviation. b) Allelochemical activity against
Rhodomomas baltica, calculated as half-effective concentration (EC50) of A. minutum cells, with 95% confidence intervals. * at limit of measured range, between 3500 and
7200 – no fit to model possible. Manuscript 4 164
Tables
Table 1 Values for physiological variables in stationary phase, defined as when µ < 0.1 d-1. All values are mean ± standard deviation of biological triplicates, with pH measurements and cell counts averaged over the stationary phase, and cellular nutrient ratios determined at the last harvesting point at the end of the experiment. (1)significantly different (p IURPDOORWKHUYDOXHVLQWKLVFROXPQDVGHWHUPLQHGE\6WXGHQW¶Vt-test on ln- transformed data (p . (2)significantly different from all other values in this column according to Welch’s t-test (unequal variances t-test, p
0.05).
Treatment Cells L-1 pH C:N N:P C:P Control 23.7 ± 5.6 (1) 9.10 ± 0.13 5.5 ± 0.4 (2) 8.5 ± 2.2 45.9 ± 10.5 P-limited 13.7 ± 1.8 (1) 9.01 ± 0.21 9.9 ± 0.8 (2) 44.6 ± 10.8 (2) 432.4 ± 86.6 (2) N-limited 9.6 ± 1.1 (1) 8.82 ± 0.24 19.9 ± 2.7 (2) 2.6 ± 0.4 50.4 ± 6.8 Manuscript 4 165
Table 2a Numbers of genes differentially expressed between treatments at transition to stationary phase and when compared to exponentially growing control cultures.
transition to stationary phase Control culture N-limited P-limited N-limited 430 - 13 transition to stationary phase P-limited 6 13 - exponential phase Control culture 6 126 21
Table 2b Numbers of genes differentially expressed between treatments in stationary phase and when compared to exponentially growing control cultures.
stationary phase Control culture N-limited P-limited stationary phase N-limited 143 - 19 P-limited 77 19 - exponential phase Control culture 1565 778 764 Manuscript 4 166
Table 3 Numbers of differentially expressed genes (SAM-based q-value <1% and fold-change 1.5) showing the same trend in several comparisons. Stationary phase: Significantly up- or down-regulated in all stationary-phase samples in relation to the control culture in exponential phase; N- / P-limitation: differentially expressed and showing the same trend between stationary-phase N- or P- limited cultures and both exponentially growing and stationary-phase control samples; nutrient limitation: identified for N- and P-limitation and same trend in both; stationary phase – control: differentially expressed between stationary and exponentially growing control and stationary phase control and both stationary- phase N- and P-limited cultures; transition to stationary phase: differentially expressed and showing the same trend in all comparisons between Day
5 samples and the control cultures in exponential phase.
Stationary phase N-limitation P-limitation nutrient limitation stationary phase - transition to
control stationary phase
down up down up down up down up down up down up
197 292 26 35 3 19 2 3 8 0 5 0 Manuscript 4 167
Table 4 Selection of genes identified as differentially expressed in all stationary-phase regimes when compared to control in exponential growth
Contig name Stationary Gene product Function vs exponential control Canonical name Control P-limited N-limited Amin_09g12r -2.91 -2.33 -3.65 ABC (ATP-binding-cassette) transporter protein transport Amin_15e01r -2.98 -2.62 -2.78 calcium-activated potassium channel transport Amin_51h24f -3.74 -2.66 -3.31 putative sugar transporter family protein transport Amin_07f04r -2.38 -2.76 -2.50 calcium/calmodulin-dependent protein kinase intracellular signalling Amin_08d07f -9.27 -5.28 -6.98 putative intracellular signaling protein intracellular signalling Amin_42g08r -3.74 -4.11 -3.93 putative mitochondrial protein, PRR repeat-containing organellar RNA-binding protein (putative) Amin_12a02r -4.38 -2.79 -2.12 60S ribosomal protein L10a. organellar translation Amin_34c10r -1.99 -2.42 -6.07 Photosystem I P700 chlorophyll a apoprotein A1 chloroplast Amin_26m06r -1.82 -2.05 -6.43 Photosystem II D2 protein chloroplast Amin_06c10r -3.82 -4.22 -4.59 malate dehydrogenase Citric acid cycle Amin_57n13r -5.78 -8.05 -23.75 cytochrome b mitochondrial Amin_38k24f -1.75 -2.11 -8.58 cytochrome c oxidase polypeptide I mitochondrial Amin_52g07r -1.69 -2.42 -3.87 DNA-3-methyladenine glycosylase DNA repair Amin_26g08f2 -2.17 -2.04 -2.29 pre-mRNA-processing-splicing factor 8, C-terminal domain Splicing Amin_34h03r -1.88 -2.15 -2.33 putative small nuclear ribonucleoprotein polypeptide E Splicing Amin_33f12f -6.92 -9.07 -9.81 18S rRNA translation Amin_44f03f -3.72 -2.41 -2.01 40S ribosomal protein S11. translation Amin_06b02r -3.02 -2.94 -2.71 40S ribosomal protein S15 translation Amin_83a11r -3.81 -2.35 -2.18 40S ribosomal protein S16. translation Amin_03c10r -3.34 -2.50 -2.15 40S ribosomal protein S4 translation Amin_57f03r -3.99 -3.10 -2.56 40S ribosomal protein S7 translation Amin_90e06f -10.52 -7.02 -6.06 40S ribosomal protein S7. translation Amin_48e09r -1.94 -2.70 -3.48 50S ribosomal protein L14, mitochondrial or chloroplast translation Amin_14a02r -5.53 -7.67 -6.25 60S ribosomal protein L12. translation Manuscript 4 168
Amin_24d11f2 -4.33 -3.65 -2.89 60S ribosomal protein L6 translation Amin_13g03r -4.49 -3.24 -2.21 60S ribosomal protein L6. translation Amin_78c12f -4.21 -2.76 -2.80 60S ribosomal protein L7a translation Amin_26m14r -8.93 -15.02 -16.98 ribosomal operon external transcribed spacer translation Amin_75d07r -4.67 -3.08 -2.64 ribosomal protein S13 translation Amin_36k19f -4.41 -3.29 -3.43 translation elongation factor-like protein translation Amin_07g02r -3.07 -2.21 -2.52 tRNA (guanine-N1-)-methyltransferase translation Amin_95c08r -4.34 -2.26 -3.27 sialyltransferase involved in protein glycosylation protein glycosylation Amin_95c05r -1.59 -2.15 -2.34 probable E3 ubiquitin-protein ligase, HECT domain-containing protein degradation Amin_11c08r -2.76 -3.36 -3.70 aspartyl proteinase family protein protein degradation
Amin_24h08f2 1.53 1.75 1.77 ABC-transporter protein transport Amin_13d06r 3.76 3.59 3.27 ABC-transporter family protein transport Amin_32d04f 2.18 2.18 2.27 ion channel similar to voltage-gated cation channels transport Amin_93m06f 1.61 2.40 1.91 TPT transporter family protein transport Amin_68f12f 2.97 2.66 2.66 inorganic H+ pyrophosphatase, vacuolar-type intracellular pH regulation Amin_08d12r 2.19 3.48 4.19 vacuolar ATP synthase subunit B intracellular pH regulation Amin_03h02f 2.18 2.33 3.96 14-3-3 protein intracellular signalling Amin_84i17r 3.13 2.40 5.91 3'5'-cyclic nucleotide phosphodiesterase family member intracellular signalling Amin_56m09r 2.12 2.00 2.19 calmodulin-like protein intracellular signalling Amin_49d23r 2.25 2.00 1.86 cGMP-dependent protein kinase intracellular signalling Amin_52f11f 1.60 2.05 2.04 dual specificity phosphatase intracellular signalling Amin_98e10f 2.06 2.53 2.47 predicted Traf-like protein intracellular signalling Amin_01h11r 3.94 3.05 6.62 protein kinase similar to shaggy-related protein kinases intracellular signalling Amin_88g07r2 3.54 2.85 3.53 protein kinase, putatively calcium-dependent intracellular signalling Amin_07c10r 6.15 5.15 6.04 Ras small GTPase, Rab type, probably involved in vesicle intracellular signalling trafficking Amin_06f11f 1.59 2.42 2.65 serine/threonine-protein kinase intracellular signalling Amin_09a03r 3.11 2.70 2.76 serine/threonine-protein phosphatase intracellular signalling Amin_68g05r 3.86 5.22 6.06 pentatricopeptide (PPR) repeat-containing protein organellar RNA-binding protein (putative) Amin_84m07f 4.21 2.42 4.20 pentatricopeptide (PPR) repeat-containing protein organellar RNA-binding protein (putative) Amin_34g06f 2.61 4.45 4.10 putative PPR repeat protein organellar RNA-binding protein Manuscript 4 169
(putative) Amin_64f10f 2.36 3.33 6.73 caroteno-chlorophyll a-c-binding protein chloroplast Amin_78d01f 6.17 4.41 4.83 light-harvesting chlorophyll a-c binding protein chloroplast Amin_41p17r 2.87 2.87 5.50 light-harvesting chlorophyll a-c binding protein chloroplast Amin_06b05r 1.80 2.00 2.63 phosphofructokinase family protein chloroplast Amin_61f12r 2.23 3.02 2.55 fumarate hydratase, putative mitochondeial Amin_07a03r 2.28 1.92 1.98 Arp2/3 complex, subunit 2 (p34-Arc) cytoskeleton Amin_21f05r 2.05 2.21 3.03 actin cytoskeleton Amin_09e07f 2.69 2.95 2.66 C-terminal motor kinesin cytoskeleton Amin_74g08f 3.59 5.16 4.85 dynein heavy chain family protein cytoskeleton Amin_07f07r 1.76 1.96 4.48 LisH domain-containing protein cytoskeleton Amin_55d08r 2.05 2.16 2.51 putative myosin, N-terminal WD 40-repeats cytoskeleton Amin_06h09r 2.54 2.36 2.19 ribonuclease HII putatively reverse transcription Amin_58c02r 2.81 3.45 3.74 putative reverse transcriptase reverse transcriptase Amin_09f06f 1.71 1.96 2.08 amine oxidase amine metabolism Amin_33f09r 2.59 2.97 3.45 putative D-3-phosphoglycerate dehydrogenase amino acid biosynthesis Amin_77b01r 3.53 2.93 3.16 putative sterol 3-beta-glucosyltransferase , partial sequence sterol modification Amin_60e01f 2.02 4.96 3.30 glycoside hydrolase family 28 family member glycoside hydrolase Amin_47c09f 3.42 2.87 2.67 adenylosuccinate lyase nucleotide metabolism Amin_83d02r2 2.87 2.25 2.15 guanine deaminase nucleotide metabolism Amin_46e08r 4.05 4.23 2.56 putative Uridine-binding protein nucleotide metabolism Amin_08h02r 1.79 2.11 2.16 DNA-directed RNA polymerases I, II, and III subunit RPABC3 transcription Amin_03c05r 2.17 2.47 3.81 ribosomal operon external transcribed spacer translation Amin_44h09f 2.46 2.97 3.13 glycyl-tRNA synthetase translation - tRNA-related Amin_69g10f 1.74 2.73 2.00 phenylalanine-tRNA synthetase translation - tRNA-related Amin_74a06r 1.82 2.09 2.19 tRNA-dihydrouridine synthase 3-like translation - tRNA-related Amin_08c10f 2.74 2.72 3.19 peptidylprolyl isomerase protein folding Amin_46g06r 2.42 2.22 2.35 diphthine synthase protein modification Amin_07g04f 2.07 1.94 2.47 aspartic protease protein degradation Amin_07g03r 2.21 2.36 2.44 proteasome subunit alpha protein degradation Amin_42o14f 1.51 2.35 1.90 ubiquitin-specific protease, putative protein degradation Manuscript 4 170
Table 5 Genes in stationary phase control cultures which were down-regulated relative to both exponential-phase and nutrient-limited cultures.
Contig name Control stationary phase vs Stationary vs stationary Gene product Control exponential
N-limited / P-limited / Control Control Amin_17h12f -6.50 16.60 7.04 hypothetical protein similar to subtilase family peptidases Amin_09f12f -5.93 5.47 2.58 putative aminopeptidase Amin_07e10f -5.13 5.97 8.56 hypothetical protein similar to alcohol dehydrogenase Amin_12a02r -4.38 2.07 1.57 60S ribosomal protein L10a Amin_64e04r -4.04 2.44 1.89 60S ribosomal protein L22 Amin_61a05r -2.95 1.77 1.99 60S ribosomal protein L21 Amin_07h11f -4.52 3.10 2.48 hypothetical protein Amin_54e08r -2.67 2.38 1.85 hypothetical protein Manuscript 4 171
Table 6 Selection of genes identified as differentially expressed in stationary-phase nutrient-limited cultures when compared to control cultures in both exponential and stationary growth phase
Contig name N-limitation P-limitation Stationary vs exponential control Stationary vs stationary Gene product
N-limited / P-limited / P-limited / Control P-limited N-limited Control Control N-limited Amin_57f07r down -3.31 -3.21 sodium-glucose cotransporter Amin_60e01f up 2.02 4.96 3.30 2.51 glycoside hydrolase family 28 family member Amin_69g10f up 1.74 2.73 2.00 1.57 phenylalanine-tRNA synthetase Amin_37h11f up 2.08 2.67 1.54 putative NALP-related protein similar to NOD3 Amin_53g06f down -1.85 -1.81 aldo-keto reductase family protein Amin_57n13r down -5.78 -8.05 -23.75 -4.22 2.96 cytochrome b Amin_14a08f down -3.14 -2.39 3.02 cytochrome b6. Amin_04f11f down -2.35 -10.25 -4.76 5.85 cytochrome b6-f complex subunit 4 Amin_38k24f down -1.75 -2.11 -8.58 -4.34 cytochrome c oxidase polypeptide I Amin_34c10r down -1.99 -2.42 -6.07 -3.06 Photosystem I P700 chlorophyll a apoprotein A1 Amin_89e05r down -2.26 -6.28 -3.48 2.97 Photosystem I P700 chlorophyll a apoprotein A1 Amin_26m06r down -1.82 -2.05 -6.43 -3.50 3.05 Photosystem II D2 protein Amin_46g07r down -2.43 -9.71 -5.69 photosystem Q(B) protein Amin_83b01r down -2.23 -4.26 -3.47 putative esterase Amin_64f10f up 2.36 3.33 6.73 2.62 caroteno-chlorophyll a-c-binding protein Amin_41p17r up 2.87 2.87 5.50 1.74 light-harvesting chlorophyll a-c binding protein Amin_98b11f up 1.90 3.92 1.93 -2.94 ribulose-bisphosphate carboxylase form II hypothetical protein similar to metal-dependent Amin_11a02f up 2.35 3.62 6.69 2.94 hydrolase ElsH hypothetical protein similar to sodium channel Amin_18f03f up 2.50 2.39 proteins Amin_100c02r up 2.03 2.70 2.18 major intrinsic protein, putative aquaporin Manuscript 4 172
Amin_71d02r up 1.72 2.88 1.68 -2.77 -4.57 pre-mRNA-processing factor Amin_36g07r up 1.51 2.61 1.70 skp1 family protein 3'5'-cyclic nucleotide phosphodiesterase family Amin_84i17r up 3.13 2.40 5.91 1.84 -2.48 member Amin_95f08r up 1.96 1.58 ADP-ribosylation factor-like protein 3 Amin_71f07r up 1.92 1.98 vWF domain-containing protein Manuscript 4 173
Table 7 Complete list of genes differentially expressed between P-limited and N-limited cultures
Contig name Stationary vs exponential control Stationary vs stationary Gene product N-limited / P-limited / P-limited / N- Control P-limited N-limited Control Control limited Amin_71d02r 1.72 2.88 1.68 -2.77 -4.57 pre-mRNA-processing factor 3'5'-cyclic nucleotide phosphodiesterase family Amin_84i17r 3.13 2.40 5.91 1.84 -2.48 member Amin_97h03r 1.64 2.73 -2.39 high-affinity nitrate transporter, putative Amin_95g04f -2.76 -3.36 2.73 3.39 ubiquitin conjugation factor E4 B
Amin_98b11f 1.90 3.92 1.93 -2.94 ribulose-bisphosphate carboxylase form II Amin_57n13r -5.78 -8.05 -23.75 -4.22 2.96 cytochrome b Amin_89e05r -2.26 -6.28 -3.48 2.97 Photosystem I P700 chlorophyll a apoprotein A1 Amin_14a08f -3.14 -2.39 3.02 cytochrome b6 Amin_26m06r -1.82 -2.05 -6.43 -3.50 3.05 Photosystem II D2 protein Amin_04f11f -2.35 -10.25 -4.76 5.85 cytochrome b6-f complex subunit 4
Amin_09e10r -4.46 hypothetical protein Amin_60e06r -1.88 -2.28 2.13 -2.60 hypothetical protein Amin_78e07r -1.72 2.16 hypothetical protein Amin_93o20f -4.47 -6.92 -18.07 -4.17 2.68 hypothetical protein Amin_22d03r -2.19 2.79 hypothetical protein Amin_42m14r 2.69 6.78 2.46 2.51 2.82 hypothetical protein Amin_66a07f -2.13 -2.25 -6.69 -3.33 2.99 hypothetical protein Amin_25j23r -5.59 -3.87 3.32 hypothetical protein Amin_21a03r 3.37 -2.06 -2.72 2.58 7.08 hypothetical protein 174
Additional files
Supplementary Table 1: Complete list of differentially expressed genes showing the same trend in several comparisons (see file)
Empty expression data cells = no significant expression difference (cutoffs: SAM-based q-value <1% and fold-change 1.5). Includes differential expression pattern across all comparisons, expression patterns found in other studies, as well as results of manual and automated annotation.
Supplementary Table 2: Carbonic anhydrases differentially expressed between stationary phase and exponential phase in control cultures (see file)
Empty expression data cells = no significant expression difference (cutoffs: SAM-based q-value <1% and fold-change 1.5). Includes differential expression pattern across all comparisons as well as results of manual and automated annotation. Synthesis 175
9 Synthesis
9.1 Characterisation of the A. minutum transcriptome
The dinoflagellate genome is highly unusual and little understood (Hackett et al., 2004;
Moreno Díaz de la Espina et al., 2005; McEwan et al., 2008), and the same is true for their gene structure and regulation (Bachvaroff & Place, 2008; Monroe & Van Dolah,
2008; Zhang & Lin, 2009). Dinoflagellates contain excessively large gene families
(Machabée et al., 1994; Sharples et al., 1996) with high intra-family sequence diversity
(Reichman et al., 2003) and a frequent occurrence of pseudogenes (Bachvaroff & Place,
2008). While no information on their gene content exists, a combination of large genome sizes (LaJeunesse et al., 2005) with an unknown but apparently dominant share of structural DNA (Sigge, 1984; Levi-Setti et al., 2008) results in an exceedingly low density of genes within the genomic sequences (McEwan et al., 2008). In order to circumvent these complications, most projects aimed at the large-scale discovery and characterisation of dinoflagellate nuclear genes are based on EST libraries (Bachvaroff et al., 2004; Tanikawa et al., 2004; Hackett et al., 2005; Uribe et al., 2008), which allow an overview over the genes represented in the transcriptome at the moment of sampling.
In order to use this approach to detect the highest possible number of genes in A. minutum, the EST library introduced in Manuscript 1 was based on pooled RNA samples from 10 different physiological treatments and 7 different timepoints over the light – dark-cycle, and the library was normalised to boost the relative abundance of less highly expressed mRNAs. Partial sequencing of 9585 cDNA library inserts (mostly sequenced from both sides, yielding 15 703 read sequences) resulted in detection of
4320 contigs, or putative genes. Of these, 28% could be automatically annotated, which is in the usual range for dinoflagellate EST libraries (John et al., 2004; Jaeckisch et al., Synthesis 176
2008). The two largest functional categories among the genes annotated to KOG categories were signal transduction mechanisms and the functional complex of posttranslational modification, protein turnover and chaperones. This might be an indication of the importance of these processes in dinoflagellates, but is probably also dependent on the extent of sequence conservation of the corresponding genes across large evolutionary distances.
Within this library, 9% of all contigs containing reads from more than one cDNA insert contained evidence for alternative splice variants. Alternative splicing is a mechanism creating multiple mRNA transcripts from a single gene, which can lead to the production of different functional proteins coded by the same sequence or to non- functional mRNAs that are thus post-transcriptionally repressed (reviewed in Barbazuk et al., 2008). About 80% or the human genes and 20% of both rice and Arabidopsis genes are known to exist in different splice variants, but their discovery is greatly dependent on EST coverage of the genome (Barbazuk et al., 2008). While the existence of the typical eukaryotic splicing machinery has been shown before (Alverca et al.,
2006), this is the first report of the frequency of alternative splicing. The variants add to the transcriptomic variation originating from the existence of the non-identical dinoflagellate gene families (Reichman et al., 2003).
A better-known dinoflagellate splicing phenomenon, the dinoflagellate-specific spliced leader (Lidie & Van Dolah, 2007; Zhang et al., 2007), was recovered in about 5% of this library. As the cDNA library is enriched in mRNA 3’-ends due to technical reasons, this is no indication of its absence in the other mRNAs, but rather an indication of complete 5’-ends in the corresponding ESTs. The percentages of SL-containing sequences among the differentially expressed genes identified in manuscript 4 were in the same range as those for the whole library. 4.3% for genes differentially expressed Synthesis 177 between any growth-limiting treatment and exponentially growing control and 5.8% for the much smaller set identified as associated with a specific treatment. This equal representation of SL sequences among the different groups of genes suggest that it is probably not restricted to a special class of constitutively transcribed multi-copy genes as has recently been suggested (Bachvaroff & Place, 2008); it is, however, in agreement with the suggestion that SL sequences can be found with all dinoflagellate mRNAs
(Zhang & Lin, 2009). This neither implies nor rules out an involvement of this sequence in the post-transcriptional regulation of gene expression, e.g. in mRNA stabilisation or in the recruitment into the actively translated mRNA pool.
• The EST library described in Manuscript 1 was composed of RNA from A. minutum
cultures subject to a variety of treatments and harvested at different circadian times.
This should ensure a high versatility of the microarray developed based on this data to
detect relevant mRNA level differences in a wide variety setups.
• The library contained indications of a rather high frequency of alternative splicing;
this is the first such report in a dinoflagellate.
• Manuscript 1 reports the proportion of identifiable dinoflagellate-specific SL
sequences in the EST library. In Manuscript 4, examination of the gene expression
patterns under different physiological conditions for involvement of the SL-
containing sequences reveals a very similar representation to that in the complete
library, suggesting that the regulation patterns for trans-spliced mRNAs are probably
not very different from those acting on the majority of sequences. Synthesis 178
9.2 Search for sequences associated with PSP toxicity
An identification of dinoflagellate genes correlated with the ability to synthesise PSP toxins would allow for the development of tools for the rapid and culture-independent discrimination of PSP-producing and non-PSP-producing Alexandrium populations, ideally independently of the exact identification of the species complex clade (John et al., 2003) or the A. minutum population of origin (Touzet et al., 2006).
The PSP toxin gene cluster of cyanobacteria has been identified (Kellmann et al.,
2008b), and its distribution and evolution among prokaryotes is now being investigated
(Kellmann et al., 2008a; Mihali et al., 2009; Moustafa et al., 2009). The second part of
Manuscript 1 describes a search for a dinoflagellate counterpart of this gene cluster. As an attempt to identify genes similar to the cyanobacterial ones lead to no result both in the A. minutum library and in other EST libraries of PSP toxin-producing Alexandrium species, an approach independent of available sequence information was chosen. As A. minutum contains closely related toxic and non-toxic clones (Montresor et al., 2004), the method of choice was a gene expression comparison between three such strains.
Two independent trials yielded 145 genes consistently higher expressed in the toxic strains, as well as eight genes not significantly expressed in the non-toxic strain. These
145 genes, and especially the 8 genes specific for the toxic strains, can be considered tentative candidates for PSP toxin-associated genes.
Switching to a very different system involving the induction of higher intracellular levels of PSP toxins, Manuscript 2 describes the response of A. minutum towards a copepod grazer. The chemical cues excreted by copepods have been shown to induce toxin production in A. minutum and to lead to up to 20x augmented intracellular toxin levels, which in turn lead to an increased resistance against grazing by these copepods.
The magnitude of this response depends on both the copepod species used and the Synthesis 179
susceptibility of the A. minutum strain (Selander et al., 2006; Bergkvist et al., 2008;
Selander et al., 2008). Using this system, a very limited set of 14 genes was shown to be
affected by copepod presence, while intracellular toxin content increased to levels 5
times higher than in the control. Among the genes up-regulated at the first day was one
of the genes that had been identified as about 10x less expressed in the non-toxic A.
minutum strain in Manuscript 1. At the third day, another sequence that had been
identified as 51x higher expressed in toxic strains was among the three differentially
expressed genes. This second sequence was not detected in a qPCR assay comparing the
toxic and non-toxic strains in manuscript 1 (see manuscript 1, Fig.4), and attempts to
amplify this sequence from cDNA and genomic DNA were only successful in the two
toxic strains (data not shown). This combination of transcriptomic comparisons between
strains of different physiological capabilities and between induced and non-induced
cultures of the same strain are a promising approach for further investigations into the
genetics of A. minutum toxin production.
• While no homologues of the cyanobacterial gene cluster could be found in the A.
minutum EST library, a microarray comparison of the genes expressed in toxic and
non-toxic strains of A. minutum lead to the identification of candidates potentially
correlated to the ability to produce toxins.
• Two of these candidates were also up-regulated in A. minutum cultures during the
induction of higher toxin cell quotas by predator cues. Synthesis 180
9.3 Transcriptome-level processes associated with growth,
nutrient status and stress response
Manuscripts 3 and 4 use the microarray probe set to investigate the transcriptome-level gene expression in A. minutum over an array of different treatments designed to study bloom-relevant conditions. As A. minutum is a coastal species that tends to bloom in relatively enclosed water bodies after nutrient input, which is often associated with freshwater runoff (Bravo et al., 2008), acclimation to different salinities should be a highly relevant feature of A. minutum biology. Manuscript 3 examines the physiological and gene expression responses under high and low salinity conditions. After a variable lag-phase, growth characteristics in the two salinity treatments paralleled those of control cultures, indicating successful acclimation. However, both high and low salinity cultures showed signs of photoinhibition, suggesting differences in chloroplast efficiency at these salinities. This was corroborated by the differential expression of several chloroplast or non-assigned organellar genes, especially in high salinity, where photoinhibition was less severe.
Overall, 8.8% of the sequences tested were identified as differentially expressed at the salinities tested. However, they displayed a very moderate amplitude with a maximum fold change of 3.5 between control and treatment. No genes were regulated in parallel between both treatments, but 12 were regulated in opposite directions. This implied an overall lack of transcriptomic stress response at this stage of acclimation. 27% of the functionally annotated differentially expressed genes were involved in mRNA modification or translation-related processes, which might hint at a high prevalence of post-transcriptional regulation under high and low salinity conditions.
The set of physiological conditions examined in Manuscript 4 was targeted at the examination of growth- and limitation-associated processes. In the course of this study, Synthesis 181
41% of all sequences represented on the microarray were identified as differentially expressed between some limiting condition and exponentially growing control cultures.
Among the identified patterns of regulation were 5 genes up-regulated in each treatment at the transition between exponential growth phase and stationary phase. These might be interesting candidates to test for suitability as biomarkers for the beginning of limitation in potential field- or mesocosm studies.
Stationary phase was mainly characterised by a major down-regulation of translational apparatus, as well as an up-regulation of genes involved in intracellular signalling. 87 genes were identified as associated with N-or P-limitation; 3 of these were up- and 2 were down- regulated in both nutrient limited treatments. This is another set of potential gene expression markers which might be useful for assessing the level of nutrient limitation in a population.
In combination with the available literature on transcriptional and translational gene regulation in dinoflagellates and model eukaryotes, the data presented in manuscripts 3 and 4 suggest that dinoflagellate gene expression is modulated at both the transcriptomic and the translational level and that the relative importance of these different mechanisms might be highly treatment specific. Synthesis 182
• While limited sets of mRNAs are differentially expressed between A. minutum
cultures acclimatised to different salinities, other physiological factors such as
nutrient limitation or culture growth phase lead to substantial alterations of the
transcriptome.
• Manuscript 4 identifies 5 genes consistently up-regulated at the transition between
exponential growth and stationary phase, which might be useful indicators for the
onset of limitation.
• 87 genes were consistently associated with either N- or P-limitation and might thus be
useful to identify the nutrient status of an A. minutum population.
9.4 Conclusion
This thesis contains the most comprehensive study of the interplay between physiological factors and gene expression in the genus Alexandrium to date. The A. minutum transcriptome was characterised on the basis of an EST library, which was designed to include the highest possible diversity of cDNA sequences. Automated annotation of the of contigs produced from this library revealed a high percentage of the functionally annotatable genes to be involved in either signal transduction or the functional category combining posttranslational modification, protein turnover and chaperones. The library allowed for the first report on the frequency of alternative splicing in dinoflagellates. An analysis of another splicing phenomenon, the dinoflagellate-specific spliced leader transsplicing, contributed new data to the current discussion on the regulation of dinoflagellate gene expression. A comparison of the prevalence of spliced leader sequences in the whole library with that in different groups of differentially expressed genes revealed no significant difference, supporting the Synthesis 183 suggestion that SL transsplicing is a general feature of dinoflagellate mRNA processing and not restricted to certain groups of genes with distinct expression patterns.
The PSP toxin genes known from cyanobacteria could not be found in A. minutum, nor in EST libraries of other PSP toxin-producing dinoflagellates. This lead to the suggestion that the corresponding dinoflagellate genes might be too different from their cyanobacterial counterparts to be identified based on sequence similarity. A comparison between toxic and non-toxic A. minutum strains revealed a number of tentative candidates for PSP toxin-associated genes. While some of the genes differentially expressed between toxic and non-toxic strains were apparently involved in gene expression-related processes or in cell cycle control, others could only be recognised as members of certain functional groups. In spite of annotations as “hydrolytic enzyme” or
“Amino-transferase”, these genes could not be assigned to biochemical reactions or pathways. The function of the majority of the genes differentially expressed between toxic and non-toxic strains could not be determined due to a lack of similarity to known sequences. Two of the sequences identified as associated with toxin-producing strains were also involved in the very specific gene expression response to a copepod grazer, which was associated with a considerable increase in PSP toxin content. These two sequences can be considered very strong candidates for genes involved in PSP toxin production or regulation.
An examination of the A. minutum gene expression response to a variety of physiological stress factors revealed very different magnitudes of transcriptome-level changes associated with the different stimuli. Acclimatisation to extreme values of high and low salinity was associated with the differential expression of a small number of genes, and amplitudes of gene expression change were moderate. While no genes were regulated in parallel, some were regulated in opposite directions in high- and low- Synthesis 184 salinity cultures. A set of gene expression comparisons between different culture growth stages and between nutrient-replete and nutrient-limited batch cultures revealed nearly half of all sequences tested to be differentially expressed between some limiting condition an the exponentially growing control. A subsequent series of cross- comparisons between the different responses identified narrow patterns associated with specific physiological conditions such as cessation of growth or nutrient limitation, identifying potential biomarkers for several physiological factors related to bloom development. Future Perspectives 185
10 Future Perspectives
In the course of this thesis, I identified a number of genes associated with specific physiological conditions. These genes are promising markers for the occurrence of the respective conditions in laboratory experiments or mesocosms. If they are to be established as strain-independent markers for the physiological state of A. minutum, their validity in different strains should be tested. These comparisons might yield universally valid cut-offs for gene expression under certain physiological conditions, possibly regarding the ratios of several specific up- and down-regulated genes in the same sample, or in relation to reference genes. This would reduce the need for measuring expression of these marker genes in relation to control samples from the same strain, enabling examination of samples containing unknown proportions of several strains, such as after several days of growth in a mixed culture or in a natural population. Such universal cut-offs would allow the development of new tools for the prediction of Alexandrium bloom probability based on the physiological status of a low- density population.
The gene expression patterns associated with the induction of higher toxin cell quotas in
A. minutum allow not only for the unusually specific study of physiological and transcriptomal changes associated with the built-up and maintenance of higher toxin levels, but also for an investigation of the sensory response of Alexandrium cells to copepod presence. Further studies on the physiology and gene expression response of A. tamarense challenged with copepod cues are currently under way (Wohlrab et al, in prep).
The most interesting group among the genes identified as potentially involved in toxicity are certainly the two genes for which both higher expression in toxin-producing strains and as up-regulation under copepod-induced higher toxin content was shown. Future Perspectives 186
The other 145 sequences identified as higher expressed in toxin-producing strains and especially the eight genes identified as specific for toxin producers also clearly warrant further investigation. These sequences could be further tested for their distribution and expression among additional toxin-producing and non-toxin-producing strains, ideally taking into account the newest available data on genetic similarity within A. minutum
(e.g. McCauley et al., 2009) to combine the few available non-toxin producers with the closest-related available toxin-producers and to include a range of distantly related toxin-producing strains.
If toxin-related gene expression patterns prove universal within A. minutum, their distribution in other PSP toxin-producing Alexandrium species such as A. tamarense and PSP toxin-producing and spirolide-producing A. ostenfeldii, as well as in the other dinoflagellate PSP toxin producers, may be of interest. Comparing the expression of dinoflagellate genes robustly associated with toxicity but not directly involved in the biosynthesis pathway with gene expression patterns in PSP toxin-producing cyanobacteria might yield common patterns of regulation or of interaction with other biochemical pathways.
EST libraries like the one the transcriptomic data of this thesis are based on typically contain only a subset of the investigated transcriptome, hence the A. minutum library is unlikely to contain the full set of genes involved in the PSP toxin synthesis pathway.
Nevertheless, given the conspicuous lack of evidence for close homologs of the cyanobacterial gene cluster in dinoflagellates to date, concentrating on de novo detection and characterisation of toxin production-related genes in Alexandrium appears more promising then searching for sequences related to the cyanobacterial genes.
Depending on the similarity of the cyanobacterial and dinoflagellate pathways and the level of homology between the corresponding genes, this might or might not apply after Future Perspectives 187 the first dinoflagellate PSP toxin synthesis genes have been confirmed and characterised.
The sequences for which the pattern of higher expression in toxic A. minutum strains persist should be further examined for their whole-transcript sequence, inter-and intra- strain variability between homologous mRNAs, genomic context of the corresponding genes, genomic copy number and organisation of their gene families. If this characterisation leads to a hypothesis on their biochemical function or a specific regulatory role in the toxin-synthesis pathway, further characterisation could be facilitated by targeted production of the proteins using heterologous expression of the corresponding gene. Such an expression of dinoflagellate proteins in both Escherichia coli and yeast has been successfully used in the characterisation of Lingulodinium polyedrum (as “Gonyaulax polyedra”) luciferase and cyclin enzymes, respectively (Bae
& Hastings, 1994; Bertomeu & Morse, 2004). This method tends to greatly simplify isolation of protein in question for direct verification of its function.
If knowledge of the whole mRNA sequences of reconfirmed candidate genes does not lead to a testable hypothesis on their function, further investigations might only be possible after establishing procedures for targeted gene inactivation in dinoflagellates.
One such method that is routinely used for the ciliate Paramecium tetraurelia, another alveolate with highly peculiar genome arrangements and extremely high gene copy numbers in the somatic nucleus, is gene silencing. In P. tetraurelia, both transformation with high-copy untranslatable but transcribable transgenes or feeding with bacteria producing double-stranded RNAs leads to post-transcriptional silencing of homologous mRNAs (Ruiz et al., 1998; Galvani & Sperling, 2002; Lepère et al., 2009). This effect has been shown to operate on subfamilies with up to about 25% sequence difference on the nucleotide level, but not between subfamilies (Ruiz et al., 1998), and so should be Future Perspectives 188 applicable to the extensive gene families of dinoflagellates. As A. minutum has been shown to ingest small prey cells when grown under low light (Jeong et al., 2005a), gene silencing by feeding might be applicable if ingestion of prey can be maximised.
Transformation of dinoflagellate cells has been demonstrated (ten Lohuis & Miller,
1998) but has not yet been adapted for thick-walled species such as A. minutum.
Gene expression studies such as the ones used in this thesis link expressed genes to physiological processes. If the studied organism is well understood and the approximate function of many expressed genes is known, then gene expression patterns can be used to examine the response of various biochemical pathways and their regulation (e.g.
Rossouw & Bauer, 2009). In less well-studied organisms such as A. minutum, in which few genes can be readily annotated based on similar sequences from better-studied organisms, patterns of gene expression can reveal association with physiological parameters. This can help to more precisely define the physiological function of genes annotated to broad functional categories, and can be a first step towards functional characterisation of genes which are newly discovered and have not yet been annotated.
Identified gene expression signatures for key physiological processes such as growth or nutrient limitation can be used to assess the physiological status of a population, e.g. when investigating the metatranscriptome of a bloom or potentially in new phytoplankton monitoring applications. Future Perspectives 189
References
ADOLF, J. E., KRUPATKINA, D., BACHVAROFF, T., PLACE, A. R. (2007). Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum. Harmful Algae 6(3): 400-412.
ALVERCA, E., FRANCA, S., MORENO DÍAZ DE LA ESPINA, S. (2006). Topology of splicing and snRNP biogenesis in dinoflagellate nuclei. Biology Of The Cell 98(12): 709-720.
ANDERSON, P. A. V., ROBERTS-MISTERLY, J., GREENBERG, R. M. (2005). The evolution of voltage-gated sodium channels: were algal toxins involved? Harmful Algae 4(1): 95-107.
BACHVAROFF, T. R., CONCEPCION, G. T., ROGERS, C. R., HERMAN, E. M., DELWICHE, C. F. (2004). Dinoflagellate expressed sequence tag data indicate massive transfer to the nuclear genome of chloroplast genes. Protist 155(1): 65-78.
BACHVAROFF, T. R., SANCHEZ-PUERTA, M. V., DELWICHE, C. F. (2005). Chlorophyll c- containing plastid relationships based on analyses of a multigene data set with all four chromalveolate lineages. Molecular Biology And Evolution 22(9): 1772-1782.
BACHVAROFF, T. R., PLACE, A. R. (2008). From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae. PLoS ONE 3(8).
BACHVAROFF, T. R., PLACE, A. R., COATS, D. W. (2009). Expressed Sequence Tags from Amoebophrya sp infecting Karlodinium veneficum: Comparing host and parasite sequences. Journal Of Eukaryotic Microbiology 56(6): 531-541.
BAE, Y. M., HASTINGS, J. W. (1994). Cloning, sequencing and expression of dinoflagellate luciferase DNA from a marine alga, Gonyaulax polyedra. Biochimica et Biophysica Acta 1219(2): 449-456.
BAKER, T. R., DOUCETTE, G. J., POWELL, C. L., BOYER, G. L., PLUMLEY, F. G. (2003). GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp. Toxicon 41(3): 339-347.
BARBAZUK, W. B., FU, Y., MCGINNIS, K. M. (2008). Genome-wide analyses of alternative splicing in plants: Opportunities and challenges. Genome Research 18(9): 1381-1392.
BERGKVIST, J., SELANDER, E., PAVIA, H. (2008). Induction of toxin production in dinoflagellates: the grazer makes a difference. Oecologia 156(1): 147-154.
BERTOMEU, T., MORSE, D. (2004). Isolation of a dinoflagellate mitotic cyclin by functional complementation in yeast. Biochemical And Biophysical Research Communications 323(4): 1172-1183.
%2'<à $ 67,//(5 - : 0$&.,(:,&= 3 &KURPDOYHRODWH SODVWLGV GLUHFW GHVFHQW RU multiple endosymbioses? Trends in Ecology & Evolution 24(3): 119-121.
BRAVO, I., VILA, M., MASÓ, M., FIGUEROA, R. I., RAMILO, I. (2008). Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches. Harmful Algae 7(4): 515.
BURKI, F., SHALCHIAN-TABRIZI, K., PAWLOWSKI, J. (2008). Phylogenomics reveals a new 'megagroup' including most photosynthetic eukaryotes. Biology Letters 4(4): 366-369.
CALADO, A. J., CRAVEIRO, S. C., MOESTRUP, Ø. (1998). Taxonomy and ultrastructure of a freshwater, heterotrophic Amphidinium (Dinophyceae) that feeds on unicellular protists. Journal of Phycology 34(3): 536-554. Future Perspectives 190
CAVALIER-SMITH, T. (2003). Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae). Philosophical Transactions of the Royal Society B-Biological Sciences 358(1429): 109-133.
CEMBELLA, A. D. (1998). Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. In Physiological Ecology of Harmful Algal Blooms (D. M. ANDERSON, A. D. CEMBELLA, G. M. HALLEGRAEFF editors), pp. 381-403. Springer Verlag, Berlin, Heidelberg, New York.
CEMBELLA, A. D., JOHN, U. (2006). Molecular physiology of toxin production and growth regulation in harmful algae. In Ecology of Harmful Algae (E. GRANÉLI , J. T. TURNER editors), pp. 215-227. Springer Berlin Heidelberg.
CHAN, Y. H., KWOK, A. C. M., TSANG, J. S. H., WONG, J. T. Y. (2006). Alveolata histone-like proteins have different evolutionary origins. Journal of Evolutionary Biology 19(5): 1717-1721.
CHANG, F. H., ANDERSON, D. M., KULIS, D. M., TILL, D. G. (1997). Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand. Toxicon 35: 393-409.
CHOU, H. N., CHEN, Y. M., CHEN, C. Y. (2004). Variety of PSP toxins in four culture strains of Alexandrium minutum collected from southern Taiwan. Toxicon 43(3): 337-340.
DODGE, J. D. (1983). Dinoflagellates: Investigation and phylogenetic speculation. European Journal of Phycology 18(4): 335-356.
DOLVEN, J. K., LINDQVIST, C., ALBERT, V. A., BJØRKLUND, K. R., YUASA, T., TAKAHASHI, O., MAYAMA, S. (2007). Molecular diversity of alveolates associated with neritic North Atlantic radiolarians. Protist 158(1): 65.
DOUCETTE, G. J., CEMBELLA, A. D., MARTIN, J. L., MICHAUD, J., COLE, T. V. N., ROLLAND, R. M. (2006). Paralytic shellfish poisoning (PSP) toxins in North Atlantic right whales Eubalaena glacialis and their zooplankton prey in the Bay of Fundy, Canada. Marine Ecology Progress Series 306: 303-313.
ELBRÄCHTER, M. (1988). Life cycle of Schizochytriodinium calani nov. gen. nov. spec., a dinoflagellate parasitizing copepod eggs. Helgoland Marine Research 42(3): 593.
FALKOWSKI, P. G., KATZ, M. E., KNOLL, A. H., QUIGG, A., RAVEN, J. A., SCHOFIELD, O., TAYLOR, F. J. R. (2004). The evolution of modern eukaryotic phytoplankton. Science 305(5682): 354- 360.
FENSOME, R. A., MACRAE, R. A., MOLDOWAN, J. M., TAYLOR, F. J. R., WILLIAMS, G. L. (1996). The early Mesozoic radiation of dinoflagellates. Paleobiology 22(3): 329-338.
FISTAROL, G. O., LEGRAND, C., SELANDER, E., HUMMERT, C., STOLTE, W., GRANÉLI , E. (2004). Allelopathy in Alexandrium spp.: effect on a natural plankton community and on algal monocultures. Aquatic Microbial Ecology 35(1): 45-56.
FLYNN, K., FRANCO, J. M., FERNÁNDEZ, P., REGUERA, B., ZAPATA, M., WOOD, G., FLYNN, K. J. (1994). Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress. Marine Ecology Progress Series 111: 99-109.
GALLACHER, S., FLYNN, K. J., FRANCO, J. M., BRUEGGEMANN, E. E., HINES, H. B. (1997). Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture. Applied and Environmental Microbiology 63(1).
GALVANI, A., SPERLING, L. (2002). RNA interference by feeding in Paramecium. Trends In Genetics 18(1): 11-12. Future Perspectives 191
GARCÉS, E., VILA, M., MASÓ, M., SAMPEDRO, N., GIACOBBE, M. G., PENNA, A. (2005). Taxon- specific analysis of growth and mortality rates of harmful dinoflagellates during bloom conditions. Marine Ecoloy Progress Series 301: 67-79.
GARCÍA, C., DEL CARMEN BRAVO, M., LAGOS, M., LAGOS, N. (2004). Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords. Toxicon 43(2): 149.
GAST, R. J., CARON, D. A. (1996). Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria. Molecular Biology And Evolution 13(9): 1192-1197.
GERACI, J. R., ANDERSON, D. M., TIMPERI, R. J., STAUBIN, D. J., EARLY, G. A., PRESCOTT, J. H., MAYO, C. A. (1989). Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Canadian Journal Of Fisheries And Aquatic Sciences 46(11): 1895-1898.
GÓMEZ, F. (2005). A list of free-living dinoflagellate species in the world’s oceans. Acta botanica Croatica 64(1): 129–212.
GÓMEZ, F., LÓPEZ-GARCÍA, P., NOWACZYK, A., MOREIRA, D. (2009). The crustacean parasites Ellobiopsis Caullery, 1910 and Thalassomyces Niezabitowski, 1913 form a monophyletic divergent clade within the Alveolata. Systematic Parasitology 74(1): 65-74.
GRANÉLI, E., HANSEN, P. J. (2006). Allelopathy in harmful algae: A mechanism to compete for resources? In Ecology of harmful algae, Ecological Series (E. GRANÉLI, J. TURNER editors), pp. 189- 201. Springer, Heidelberg.
GROISILLIER, A., MASSANA, R., VALENTIN, K., VAULOTL, D., GUILLOUL, L. (2006). Genetic diversity and habitats of two enigmatic marine alveolate lineages. Aquatic Microbial Ecology 42(3): 277- 291.
GUILLOU, L., VIPREY, M., CHAMBOUVET, A., WELSH, R. M., KIRKHAM, A. R., MASSANA, R., SCANLAN, D. J., WORDEN, A. Z. (2008). Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environmental Microbiology 10(12): 3349-3365.
HACKETT, J., SCHEETZ, T., YOON, H., SOARES, M., BONALDO, M., CASAVANT, T., BHATTACHARYA, D. (2005). Insights into a dinoflagellate genome through expressed sequence tag analysis. BMC Genomics 6(1): 80.
HACKETT, J. D., ANDERSON, D. M., ERDNER, D. L., BHATTACHARYA, D. (2004). Dinoflagellates: A remarkable evolutionary experiment. American Journal of Botany 91(10): 1523-1534.
HALLEGRAEFF, G. A. (1993). A review of harmful algal blooms and their apparent global increase. Phycologia 32(2): 79-99.
HANSEN, G., DAUGBJERG, N., FRANCO, J. M. (2003). Morphology, toxin composition and LSU rDNA phylogeny of Alexandrium minutum (Dinophyceae) from Denmark, with some morphological observations on other European strains. Harmful Algae 2(4): 317-335.
HOLD, G. L., SMITH, E. A., BIRKBECK, T. H., GALLACHER, S. (2001). Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria. FEMS Microbiology Ecology 36(2-3): 223.
HOPPENRATH, M., LEANDER, B. S. (2009). Molecular phylogeny of Parvilucifera prorocentri (Alveolata, Myzozoa): Insights into perkinsid character evolution. Journal Of Eukaryotic Microbiology 56(3): 251-256.
HWANG, D. F., TSAI, Y. H. (1999). Toxins in toxic Taiwanese crabs. Food Reviews International 15(2): 145-162.
HWANG, D. F., LU, Y. H. (2000). Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum. Toxicon 38(11): 1491-1503. Future Perspectives 192
IRIGOIEN, X., FLYNN, K. J., HARRIS, R. P. (2005). Phytoplankton blooms: a "loophole" in microzooplankton grazing impact? Journal Of Plankton Research 27(4): 313-321.
ISHIDA, Y., UCHIDA, A., SAKO, Y. (1998). Genetic and biochemical approaches to PSP toxin production of toxic dinoflagellates. In Physiological Ecology of Harmful Algal Blooms (D. M. ANDERSON, A. D. CEMBELLA, G. M. HALLEGRAEFF editors), pp. 49-58. Springer-Verlag, Heidelberg.
JAECKISCH, N., GLÖCKNER, G., VOGEL, H., CEMBELLA, A., JOHN, U. (2008). Genomic characterization of the spirolide-producing dinoflagellate Alexandrium ostenfeldii with special emphasis on PKS genes. In Proceedings of 12th International Conference on Harmful Algae, Copenhagen, Denmark. 04.-08.09.2006,(Ø. MOESTRUP, G. DOUCETTE, H. ENEVOLDSEN editors)
JANOUŠKOVEC, J., HORÁK, A., OBORNÍK, M., LUKEŠ, J., KEELING, P. J. (2010). A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids. Proceedings of the National Academy of Sciences of the United States of America 107(24): 10949-10954.
JASTI, S., SIERACKI, M. E., POULTON, N. J., GIEWAT, M. W., ROONEY-VARGA, J. N. (2005). Phylogenetic diversity and specificity of bacteria closely associated with Alexandrium spp. and other phytoplankton. Applied and Environmental Microbiology 71(7): 3483-3494.
JEONG, H. J., PARK, J. Y., NHO, J. H., PARK, M. O., HA, J. H., SEONG, K. A., JENG, C., SEONG, C. N., LEE, K. Y., YIH, W. H. (2005a). Feeding by red-tide dinoflagellates on the cyanobacterium Synechococcus. Aquatic Microbial Ecology 41(2): 131-143.
JEONG, H. J., YOO, Y. D., PARK, J. Y., SONG, J. Y., KIM, S. T., LEE, S. H., KIM, K. Y., YIH, W. H. (2005b). Feeding by phototrophic red-tide dinoflagellates: five species newly revealed and six species previously known to be mixotrophic Aquatic Microbial Ecology 40(2): 133-150.
JOHN, U., FENSOME, R. A., MEDLIN, L. K. (2003). The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distributions within the Alexandrium tamarense "species complex" (Dinophyceae). Molecular Biology And Evolution 20(7): 1015-1027.
JOHN, U., MOCK, T., VALENTIN, K., CEMBELLA, A. D., MEDLIN, L. K. (2004). Dinoflagellates come from outer space but haptophytes and diatoms do not. In Proceedings of Harmful Algae 2002, St. Pete Beach, Florida, USA. 21–25.10.2002,(K. A. STEIDINGER, J. H. LANDSBERG, C. R. TOMAS, G. A. VARGO editors)
JONSSON, P. R., PAVIA, H., TOTH, G. (2009). Formation of harmful algal blooms cannot be explained by allelopathic interactions. Proceedings of the National Academy of Sciences of the United States of America 106(27): 11177-11182.
JOSEPH, S. J., FERNÁNDEZ-ROBLEDO, J. A., GARDNER, M. J., EL-SAYED, N. M., KUO, C. H., SCHOTT, E. J., WANG, H., KISSINGER, J. C., VASTA, G. R. (2010). The alveolate Perkinsus marinus: Biological insights from EST gene discovery. BMC Genomics 11.
KAO, C. Y., WALKER, S. E. (1982). Active groups of saxitoxin and tetrodotoxin as deduced from actions of saxitoxin analogues on frog muscle and squid axon. Journal of Physiology 323(FEB): 619-637.
KELLMANN, R., NEILAN, B. A. (2007). Biochemical characterization of paralytic shellfish toxin biosynthesis in vitro. Journal of Phycology 43(3): 497-508.
KELLMANN, R., MICHALI, T. K., NEILAN, B. A. (2008a). Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers. Journal of Molecular Evolution 67(5): 526-538.
KELLMANN, R., MIHALI, T. K., JEON, Y. J., PICKFORD, R., POMATI, F., NEILAN, B. A. (2008b). Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Applied and Environmental Microbiology 74(13): 4044-4053. Future Perspectives 193
KROCK, B., SEGUEL, C. G., CEMBELLA, A. D. (2007). Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry. Harmful Algae 6(5): 734-744.
LAJEUNESSE, T. C., LAMBERT, G., ANDERSEN, R. A., COFFROTH, M. A., GALBRAITH, D. W. (2005). Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. Journal of Phycology 41(4): 880-886.
LANDRY, M. R., KIRCHMAN, D. L. (2002). Microbial community structure and variability in the tropical Pacific. Deep Sea Research Part II: Topical Studies in Oceanography 49(13-14): 2669-2693.
LANDSBERG, J. H., STEIDINGER, K. A., BLAKESLEY, B. A., ZONDERVAN, R. L. (1994). Scanning electron microscope study of dinospores of Amyloodinium cf. ocellatum, a pathogenic dinoflagellate parasite of marine fish, and comments on its relationship to the Peridiniales. Diseases Of Aquatic Organisms 20(1): 23-32.
LANE, C. E., ARCHIBALD, J. M. (2008). The eukaryotic tree of life: endosymbiosis takes its TOL. Trends in Ecology & Evolution 23(5): 268-275.
LEANDER, B. S., KEELING, P. J. (2003). Morphostasis in alveolate evolution. Trends in Ecology & Evolution 18(8): 395-402.
LEANDER, B. S., KUVARDINA, O. N., ALESHIN, V. V., MYLNIKOV, A. P., KEELING, P. J. (2003). Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): Insights into the phagotrophic ancestry of apicomplexans. Journal Of Eukaryotic Microbiology 50(5): 334-340.
LEGRAND, C., RENGEFORS, K., FISTAROL, G. O., GRANÉLI, E. (2003). Allelopathy in phytoplankton - biochemical, ecological and evolutionary aspects. Phycologia 42(4): 406-419.
LEHANE, L. (2000). Paralytic Shellfish Poisoning: A review. National Office of Animal and Plant Health, Australia, Canberra.
LEPÈRE, C., DOMAIZON, I., DEBROAS, D. (2008). Unexpected importance of potential parasites in the composition of the freshwater small-eukaryote community. Applied and Environmental Microbiology 74(10): 2940-2949.
LEPÈRE, G., NOWACKI, M., SERRANO, V., GOUT, J. F., GUGLIELMI, G., DUHARCOURT, S., MEYER, E. (2009). Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia. Nucleic Acids Research 37(3): 903-915.
LEUNG, K. M. Y., DUDGEON, D. (2008). Ecological risk assessment and management of exotic organisms associated with aquaculture activities. In Understanding and applying risk analysis in aquaculture (M. G. BONDAD-REANTASO, J. R. ARTHUR, R. P. SUBASINGHE editors), FAO, Rome.
LEVI-SETTI, R., GAVRILOV, K. L., RIZZO, P. J. (2008). Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry. European Journal of Cell Biology 87(12): 963-976.
LEVINSEN, H., NIELSEN, T. G. (2002). The trophic role of marine pelagic ciliates and heterotrophic dinoflagellates in arctic and temperate coastal ecosystems: A cross-latitude comparison. Limnology and Oceanography 47(2): 427-439.
LIDIE, K. B., RYAN, J. C., BARBIER, M., VAN DOLAH, F. M. (2005). Gene expression in Florida red tide dinoflagellate Karenia brevis: Analysis of an expressed sequence tag library and development of DNA microarray. Marine Biotechnology 7(5): 481-493.
LIDIE, K. B., VAN DOLAH, F. M. (2007). Spliced leader RNA-mediated trans-splicing in a dinoflagellate, Karenia brevis. Journal Of Eukaryotic Microbiology 54(5): 427-435.
LILLY, E. L., HALANYCH, K. M., ANDERSON, D. M. (2005). Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group. Harmful Algae 4(6): 1004–1020. Future Perspectives 194
LILLY, E. L., HALANYCH, K. M., ANDERSON, D. M. (2007). Species boundaries and global biogeography of the Alexandrium tamarense complex (Dinophyceae). Journal of Phycology 43(6): 1329- 1338.
LIM, P.-T., SATO, S., VAN THUOC, C., TU, P. T., HUYEN, N. T. M., TAKATA, Y., YOSHIDA, M., KOBIYAMA, A., KOIKE, K., OGATA, T. (2007). Toxic Alexandrium minutum (Dinophyceae) from Vietnam with new gonyautoxin analogue. Harmful Algae 6(3): 321.
LIVOLANT, F., BOULIGAND, Y. (1978). New observations on the twisted arrangement of Dinoflagellate chromosomes. Chromosoma 68(1): 21.
LLEWELLYN, L. E., DODD, M. J., ROBERTSON, A., ERICSON, G., DE KONING, C., NEGRI, A. P. (2002). Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneus. Toxicon 40(10): 1463-1469.
LOMBARD, F., EREZ, J., MICHEL, E., LABEYRIE, L. (2009). Temperature effect on respiration and photosynthesis of the symbiont-bearing planktonic foraminifera Globigerinoides ruber, Orbulina universa, and Globigerinella siphonifera. Limnology and Oceanography 54(1): 210-218.
LOPES, R. M., SILVEIRA, M. (1994). Symbiosis between a pelagic flatworm and a dinoflagellate from a tropical area: structural observations. Hydrobiologia 287(3): 277.
LU, Y. H., CHAI, T. J., HWANG, D. F. (2000). Isolation of bacteria from toxic dinoflagellate Alexandrium minutum and their effects on algae toxicity. Journal of Natural Toxins 9(4): 409-417.
MA, H. Y., KROCK, B., TILLMANN, U., CEMBELLA, A. (2009). Preliminary characterization of extracellular allelochemicals of the toxic marine dinoflagellate Alexandrium tamarense using a Rhodomonas salina bioassay. Marine Drugs 7(4): 497-522.
MACHABÉE, S., WALL, L., MORSE, D. (1994). Expression and genomic organization of a dinoflagellate gene family. Plant Molecular Biology V25(1): 23.
MACKENZIE, L., BERKETT, N. (1997). Cell morphology and PSP-toxin profiles of Alexandrium minutum in the Marlborough Sounds, New Zealand. New Zealand Journal of Marine and Freshwater Research 31(3): 403-409.
MACRAE, R. A., FENSOME, R. A., WILLIAMS, G. L. (1996). Fossil dinoflagellate diversity, originations, and extinctions and their significance. Canadian Journal of Botany-Revue Canadienne De Botanique 74(11): 1687-1694.
MANGIALAJO, L., BERTOLOTTO, R., CATTANEO-VIETTI, R., CHIANTORE, M., GRILLO, C., LEMEE, R., MELCHIORRE, N., MORETTO, P., POVERO, P., RUGGIERI, N. (2008). The toxic benthic dinoflagellate Ostreopsis ovata: Quantification of proliferation along the coastline of Genoa, Italy. Marine Pollution Bulletin 56(6): 1209-1214.
MCCAULEY, L. A. R., ERDNER, D. L., NAGAI, S., RICHLEN, M. L., ANDERSON, D. M. (2009). Biogeographic analysis of the globally distributed harmful algal bloom species Alexandrium minutum (Dinophyceae) based on rRNA gene sequences and microsatellite markers. Journal of Phycology 45(2): 454-463.
MCEWAN, M., HUMAYUN, R., SLAMOVITS, C. H., KEELING, P. J. (2008). Nuclear genome sequence survey of the dinoflagellate Heterocapsa triquetra. Journal Of Eukaryotic Microbiology 55(6): 530-535.
MIHALI, T., KELLMANN, R., NEILAN, B. (2009). Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. BMC Biochemistry 10(1): 8.
MOESTRUP, Ø. (ed.) (2004). IOC Taxonomic reference list of toxic algae. Intergovernmental Oceanographic Commission of UNESCO, ioc.unesco.org/hab/data.htm. Future Perspectives 195
MONROE, E. A., VAN DOLAH, F. M. (2008). The toxic dinoflagellate Karenia brevis encodes novel type I-like polyketide synthases containing discrete catalytic domains. Protist 159(3): 471-482.
MONTRESOR, M., LOVEJOY, C., ORSINI, L., PROCACCINI, G., ROY, S. (2003). Bipolar distribution of the cyst-forming dinoflagellate Polarella glacialis. Polar Biology 26(3): 186-194.
MONTRESOR, M., JOHN, U., BERAN, A., MEDLIN, L. K. (2004). Alexandrium tamutum sp. nov. (Dinophyceae): A new nontoxic species in the genus Alexandrium. Journal of Phycology 40(2): 398-411.
MOORE, R. B., OBORNIK, M., JANOUŠKOVEC, J., CHRUDIMSKÝ, T., VANCOVÁ, M., GREEN, D. H., WRIGHT, S. W., DAVIES, N. W., BOLCH, C. J. S., HEIMANN, K., ŠLAPETA, J., HOEGH- GULDBERG, O., LOGSDON, J. M., CARTER, D. A. (2008). A photosynthetic alveolate closely related to apicomplexan parasites. Nature 451(7181): 959-963.
MOREIRA, D., LÓPEZ-GARCÍA, P. (2003). Are hydrothermal vents oases for parasitic protists? Trends in Parasitology 19(12): 556-558.
MORENO DÍAZ DE LA ESPINA, S., ALVERCA, E., CUADRADO, A., FRANCA, S. (2005). Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates. European Journal of Cell Biology 84(2-3): 137-149.
MOUSTAFA, A., LORAM, J. E., HACKETT, J. D., ANDERSON, D. M., PLUMLEY, F. G., BHATTACHARYA, D. (2009). Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS ONE 4(6): e5758.
MOUSTAFA, A., EVANS, A., KULIS, D., HACKETT , J., ERDNER, D., ANDERSON, D., BHATTACHARYA, D. (2010). Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence. PLoS ONE 5(3): e9688.
NAGAI, S., LIAN, C., YAMAGUCHI, S., HAMAGUCHI, M., MATSUYAMA, Y., ITAKURA, S., SHIMADA, H., KAGA, S., YAMAUCHI, H., SONDA, Y., NISHIKAWA, T., KIM, C. H., HOGETSU, T. (2007). Microsatellite markers reveal population genetic structure of the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) in Japanese coastal waters. Journal of Phycology 43(1): 43-54.
NASH, E. A., BARBROOK, A. C., EDWARDS-STUART, R. K., BERNHARDT, K., HOWE, C. J., NISBET, R. E. R. (2007). Organization of the mitochondrial genome in the dinoflagellate Amphidinium carterae. Molecular Biology And Evolution 24(7): 1528-1536.
OBORNÍK, M., JANOUŠKOVEC, J., CHRUDIMSKÝ, T., LUKEŠ, J. (2009). Evolution of the apicoplast and its hosts: From heterotrophy to autotrophy and back again. International Journal For Parasitology 39(1): 1-12.
OKAMOTO, O. K., HASTINGS, J. W. (2003). Novel dinoflagellate clock-related genes identified through microarray analysis. Journal of Phycology 39(3): 519-526.
OSHIMA, Y. (1995). Chemical and enzymatic transformation of paralytic shellfish toxins in marine organisms. In Harmful Marine Algal Blooms (P. LASSUS, G. ARZUL, E. ERARD, P. GENTIEN, C. MARCAILLOU editors), pp. 475-480. Lavoisier, Paris (France).
PALLIANI, R. B., BURATTI, N. (2006). High diversity dinoflagellate cyst assemblages from the Late Triassic of southern England: new information on early dinoflagellate evolution and palaeogeography. Lethaia 39(4): 305-312.
PARAB, S. G., MATONDKAR, S. G. P., GOMES, H. D., GOES, J. I. (2006). Monsoon driven changes in phytoplankton populations in the eastern Arabian Sea as revealed by microscopy and HPLC pigment analysis. Continental Shelf Research 26(20): 2538-2558.
POPOVICH, C. A., SPETTER, C. V., MARCOVECCHIO, J. E., FREIJE, R. H. (2008). Dissolved nutrient availability during winter diatom bloom in a turbid and shallow estuary (Bahía Blanca, Argentina). Journal of Coastal Research 24(1): 95-102. Future Perspectives 196
PROL, M. J., GUISANDE, C., BARREIRO, A., MÍGUEZ, B., DE LA IGLESIA, P., VILLAR, A., GAGO-MARTÍNEZ, A., COMBARRO, M. P. (2009). Evaluation of the production of paralytic shellfish poisoning toxins by extracellular bacteria isolated from the toxic dinoflagellate Alexandrium minutum. Canadian Journal of Microbiology 55(8): 943-954.
QUIGG, A., FINKEL, Z. V., IRWIN, A. J., ROSENTHAL, Y., HO, T. Y., REINFELDER, J. R., SCHOFIELD, O., MOREL, F. M. M., FALKOWSKI, P. G. (2003). The evolutionary inheritance of elemental stoichiometry in marine phytoplankton. Nature 425(6955): 291-294.
REEB, V. C., PEGLAR, M. T., YOON, H. S., BAI, J. R., WU, M., SHIU, P., GRAFENBERG, J. L., REYES-PRIETO, A., RÜMMELE, S. E., GROSS, J., BHATTACHARYA, D. (2009). Interrelationships of chromalveolates within a broadly sampled tree of photosynthetic protists. Molecular Phylogenetics And Evolution 53(1): 202-211.
REICHMAN, J. R., WILCOX, T. P., VIZE, P. D. (2003). PCP gene family in Symbiodinium from Hippopus hippopus: Low levels of concerted evolution, isoform diversity, and spectral tuning of chromophores. Molecular Biology And Evolution 20(12): 2143-2154.
REID, P. C., LANCELOT, C., GIESKES, W. W. C., HAGMEIER, E., WEICHART, G. (1990). Phytoplankton of the North Sea and its dynamics: A review. Netherlands Journal of Sea Research 26(2- 4): 295-331.
RENGEFORS, K., LEGRAND, C. (2007). Broad allelopathic activity in Peridinium aciculiferum (Dinophyceae). European Journal of Phycology 42(4): 341-349.
RIEGEL, W. (2008). The Late Palaeozoic phytoplankton blackout - Artefact or evidence of global change? Review Of Palaeobotany And Palynology 148(2-4): 73-90.
ROSSOUW, D., BAUER, F. F. (2009). Comparing the transcriptomes of wine yeast strains: toward understanding the interaction between environment and transcriptome during fermentation. Applied Microbiology and Biotechnology 84(5): 937-954.
RUIZ, F., VAYSSIÉ, L., KLOTZ, C., SPERLING, L., MADEDDU, L. (1998). Homology-dependent gene silencing in Paramecium. Molecular Biology of the Cell 9(4): 931-943.
SAKO, Y., YOSHIDA, T., UCHIDA, A., ARAKAWA, O., NOGUCHI, T., ISHIDA, Y. (2001). Purification and characterization of a sulfotransferase specific to N-21 of saxitoxin and gonyautoxin 2+3 from the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae). Journal of Phycology 37(6): 1044- 1051.
SANCHEZ-PUERTA, M. V., LIPPMEIER, J. C., APT, K. E., DELWICHE, C. F. (2007). Plastid genes in a non-photosynthetic dinoflagellate. Protist 158(1): 105.
SCHOLIN, C. A., HALLEGRAEFF, G. M., ANDERSON, D. M. (1995). Molecular evolution of the Alexandrium tamarense species complex (Dinophyceae)-dispersal in the North American and West Pacific regions. Phycologia 34(6): 472-485.
SELANDER, E., THOR, P., TOTH, G., PAVIA, H. (2006). Copepods induce paralytic shellfish toxin production in marine dinoflagellates. Proceedings Of The Royal Society B-Biological Sciences 273(1594): 1673-1680.
SELANDER, E., CERVIN, G., PAVIA, H. (2008). Effects of nitrate and phosphate on grazer-induced toxin production in Alexandrium minutum. Limnology and Oceanography 53(2): 523-530.
SHARPLES, F. P., WRENCH, P. M., OU, K., HILLER, R. G. (1996). Two distinct forms of the peridinin-chlorophyll a-protein from Amphidinium carterae. Biochimica et Biophysica Acta- Bioenergetics 1276(2): 117.
SHIMIZU, Y., NORTE, M., HORI, A., GENENAH, A., KOBAYASHI, M. (1984). Biosynthesis of saxitoxin analogs: the unexpected pathway. Journal of the American Chemical Society 106(21): 6433- 6434. Future Perspectives 197
SHIMIZU, Y. (1996). Microalgal metabolites: a new perspective. Annual Review of Microbiology 50: 431-465.
SHUMWAY, S. E., ALLEN, S. M., BOERSMA, P. D. (2003). Marine birds and harmful algal blooms: sporadic victims or under-reported events? Harmful Algae 2(1): 1-17.
SHYAM, R., SARMA, Y. (1978). Cytology of Indian freshwater Dinophyceae. Botanical Journal of the Linnean Society 76(2): 145-159.
SIGGE, D. C. (1984). Structural DNA and genetically active DNA in dinoflagellate chromosomes. Biosystems 16: 203-210.
SKOVGAARD, A., MENESES, I., ANGÉLICO, M. M. (2009). Identifying the lethal fish egg parasite Ichthyodinium chabelardi as a member of Marine Alveolate Group I. Environmental Microbiology 11(8): 2030-2041.
SLAMOVITS, C. H., KEELING, P. J. (2008). Widespread recycling of processed cDNAs in dinoflagellates. Current Biology 18(13): R550-R552.
SLUIJS, A., PROSS, J., BRINKHUIS, H. (2005). From greenhouse to icehouse; organic-walled dinoflagellate cysts as paleoenvironmental indicators in the Paleogene. Earth-Science Reviews 68(3-4): 281-315.
SMAYDA, T. J. (1990). Novel and nuisance phytoplankton blooms in the sea: Evidence for a global epidemic. In Toxic Marine Phytoplankton (E. GRANÉLI, B. SUNDSTRÖM, L. EDLER, D. M. ANDERSON editors), pp. 29-40. Elsevier, New York.
SMAYDA, T. J. (1997). What is a bloom? A commentary. Limnology and Oceanography 42(5): 1132- 1136.
SPILLING, K. (2007). Dense sub-ice bloom of dinoflagellates in the Baltic Sea, potentially limited by high pH. Journal Of Plankton Research 29(10): 895-901.
STELTER, K., EL-SAYED, N. M., SEEBER, F. (2007). The expression of a plant-type ferredoxin redox system provides molecular evidence for a plastid in the early dinoflagellate Perkinsus marinus. Protist 158(1): 119-130.
STUCKEN, K., JOHN, U., CEMBELLA, A., MURILLO, A. A., SOTO-LIEBE, K., FUENTES- VALDÉS, J. J., FRIEDEL, M., PLOMINSKY, A. M., VÁSQUEZ, M., GLÖCKNER, G. (2010). The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications. PLoS ONE 5(2): e9235.
TANG, Y. Z., GOBLER, C. J. (2009). Characterization of the toxicity of Cochlodinium polykrikoides isolates from Northeast US estuaries to finfish and shellfish. Harmful Algae 8(3): 454-462.
TANIKAWA, N., AKIMOTO, H., OGOH, K., CHUN, W., OHMIYA, Y. (2004). Expressed sequence tag analysis of the dinoflagellate Lingulodinium polyedrum during dark phase. Photochemistry and Photobiology 80(1): 31-35.
TARONCHER-OLDENBURG, G., KULIS, D. M., ANDERSON, D. M. (1999). Coupling of saxitoxin biosynthesis to the G(1) phase of the cell cycle in the dinoflagellate Alexandrium fundyense: Temperature and nutrient effects. Natural Toxins 7(5): 207-219.
TEN LOHUIS, M. R., MILLER, D. J. (1998). Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. The Plant Journal 13: 427.
THOMSON, P., MCMINN, A., KIESSLING, I., WATSON, M., GOLDSWORTHY, P. (2006). Composition and succession of dinoflagellates and chrysophytes in the upper fast ice of Davis Station, East Antarctica. Polar Biology 29(4): 337. Future Perspectives 198
TILLMANN, U., HESSE, K. J. (1998). On the quantitative importance of heterotrophic microplankton in the northern German Wadden Sea. Estuaries 21(4A): 585-596.
TILLMANN, U., JOHN, U. (2002). Toxic effects of Alexandrium spp. on heterotrophic dinoflagellates: an allelochemical defence mechanism independent of PSP-toxin content. Marine Ecology Progress Series 230: 47-58.
TILLMANN, U. (2004). Interactions between planktonic microalgae and protozoan grazers. Journal Of Eukaryotic Microbiology 51(2): 156-168.
TILLMANN, U., ALPERMANN, T., JOHN, U., CEMBELLA, A. (2008). Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists. Harmful Algae 7(1): 52-64.
TILLMANN, U., ALPERMANN, T. L., DA PURIFICAÇÃO, R. C., KROCK, B., CEMBELLA, A. (2009). Intra-population clonal variability in allelochemical potency of the toxigenic dinoflagellate Alexandrium tamarense. Harmful Algae 8(5): 759-769.
TÖBE, K. (2003). Investigations into the role of bacteria/dinoflagellate interactions in Paralytic Shellfish Poisoning. In University Bremen, p. 174. University Bremen, Bremen.
TOULZA, E., SHIN, M.-S., BLANC, G., AUDIC, S., LAABIR, M., COLLOS, Y., CLAVERIE, J.-M., GRZEBYK, D. (2010). Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella (Dinophyceae). Applied and Environmental Microbiology 76(13): 4521-4529.
TOUZET, N., FRANCO, J. M., RAINE, R. (2006). Inter- and intra-specific variability in morphogenetics and toxin composition of Alexandrium spp. in Irish coastal waters. African Journal Of Marine Science 28(2): 181-184.
TOUZET, N., FRANCO, J. M., RAINE, R. (2007). Characterization of nontoxic and toxin-producing strains of Alexandrium minutum (Dinophyceae) in Irish coastal waters. Applied and Environmental Microbiology 73(10): 3333-3342.
TRAINER, V. L., BADEN, D. G. (1999). High affinity binding of red tide neurotoxins to marine mammal brain. Aquatic Toxicology 46(2): 139-148.
TURNER, J. T., TESTER, P. A. (1997). Toxic marine phytoplankton, zooplankton grazers, and pelagic food webs. Limnology and Oceanography 42(5): 1203-1214.
URIBE, P., ESPEJO, R. T. (2003). Effect of associated bacteria on the growth and toxicity of Alexandrium catenella. Applied and Environmental Microbiology 69(1): 659-662.
URIBE, P., FUENTES, D., VALDÉS, J., SHMARYAHU, A., ZÚÑIGA, A., HOLMES, D., VALENZUELA, P. D. T. (2008). Preparation and analysis of an expressed sequence tag library from the toxic dinoflagellate Alexandrium catenella Marine Biotechnology 10(6): 692-700.
VAN DOLAH, F. M. (2000). Marine algal toxins: Origins, health effects, and their increased occurrence. Environmental Health Perspectives 108: 133-141.
VAN DOLAH, F. M., LIDIE, K. B., MOREY, J. S., BRUNELLE, S. A., RYAN, J. C., MONROE, E. A., HAYNES, B. L. (2007). Microarray analysis of diurnal- and circadian-regulated genes in the Florida red- tide dinoflagellate Karenia brevis (Dinophyceae). Journal of Phycology 43(4): 741-752.
VARGAS, C. A., MARTÍNEZ, R. A. (2009). Grazing impact of natural populations of ciliates and dinoflagellates in a river-influenced continental shelf. Aquatic Microbial Ecology 56(1): 93-108.
VILA, M., GIACOBBE, M. G., MASÓ, M., GANGEMI, E., PENNA, A., SAMPEDRO, N., AZZARO, F., CAMP, J., GALLUZZI, L. (2005). A comparative study on recurrent blooms of Alexandrium minutum in two Mediterranean coastal areas. Harmful Algae 4(4): 673-695. Future Perspectives 199
VOGELBEIN, W. K., LOVKO, V. J., SHIELDS, J. D., REECE, K. S., MASON, P. L., HAAS, L. W., WALKER, C. C. (2002). Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion. Nature 418(6901): 967-970.
VOLKMAN, J. K., KEARNEY, P., JEFFREY, S. W. (1990). A new source of 4-methyl sterols and 5[alpha](H)-stanols in sediments: prymnesiophyte microalgae of the genus Pavlova. Organic Geochemistry 15(5): 489-497.
VOLKMAN, J. K., BARRETT, S. M., DUNSTAN, G. A., JEFFREY, S. W. (1993). Geochemical significance of the occurrence of dinosterol and other 4-methyl sterols in a marine diatom. Organic Geochemistry 20(1): 7-15.
WANG, D., ZHANG, S., HONG, H. (2007). A sulfotransferase specific to N-21 of gonyautoxin 2/3 from crude enzyme extraction of toxic dinoflagellate Alexandrium tamarense CI01. Chinese Journal of Oceanology and Limnology 25(2): 227.
WEAR, R. G., GARDNER, J. P. A. (2001). Biological effects of the toxic algal bloom of February and March 1998 on the benthos of Wellington Harbour, New Zealand. Marine Ecology Progress Series 218: 63-76.
WYATT, T., JENKINSON, I. (1997). Notes on Alexandrium population dynamics. Journal Of Plankton Research 19(5): 551-575.
YANG, E. J., CHOI, J. K., HYUN, J. H. (2004). Distribution and structure of heterotrophic protist communities in the northeast equatorial Pacific Ocean. Marine Biology 146(1): 1-15.
YENTSCH, C. S., LAPOINTE, B. E., POULTON, N., PHINNEY, D. A. (2008). Anatomy of a red tide bloom off the southwest coast of Florida. Harmful Algae 7(6): 817-826.
YOSHIDA, T., SAKO, Y., UCHIDA, A., KAKUTANI, T., ARAKAWA, O., NOGUCHI, T., ISHIDA, Y. (2002). Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae). Fisheries Science 68(3): 634-642.
ZHANG, H., HOU, Y. B., MIRANDA, L., CAMPBELL, D. A., STURM, N. R., GAASTERLAND, T., LIN, S. J. (2007). Spliced leader RNA trans-splicing in dinoflagellates. Proceedings of the National Academy of Sciences of the United States of America 104(11): 4618-4623.
ZHANG, H., CAMPBELL, D. A., STURM, N. R., LIN, S. (2009). Dinoflagellate spliced leader RNA genes display a variety of sequences and genomic arrangements. Mol Biol Evol 26(8): 1757-1771.
ZHANG, H., LIN, S. (2009). Retrieval of missing spliced leader in dinoflagellates. PLoS ONE 4(1). 200
Appendix 201
I. Manuscript 1, additional file 1: Primer tables and
respective PCR conditions 202 203 204 205
II. Manuscript 1, additional file 3: Phyml-based
likelihood trees with bootstrap support values
14 phylogenies of A. minutum EST contigs that produced significant (e<10-4) BLAST hits with cyanobacterial sxt-related genes. Phylogenies were calculated including the A. minutum contig sequence, the corresponding cyanobacterial gene, and their closest
SwissProt matches as identified by PhylogGena (Top10Select-mode). Phylogenies M.2 and N.2 additionally include the best 20 hits produced by blasting the Alexandrium sequences against the NCBI non-redundant protein sequence database (nr). 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222
III. Manuscript 1, additional file 4: Rarefaction curve
Generated ESTs and assembled cluster as contigs were analysed using http://www.biology.ualberta.ca/jbrzusto/rarefact.php#Calculator. 223 224
IV. Manuscript 2, Supplementary table: information on the
genes up-regulated at Day 1 or Day 3 of copepod-
induced PSP toxin production
Log2 values of fold change exposed / control (mean of triplicates), standard deviation of log2 values, fold change exposed / control (geometric mean of triplicates), annotation or lack of annotation confirmed manually. Geometric mean toxic strains / non-toxic strains
(Manuskript 1). With value of best BLAST hit against other dinoflagellate EST libraries, NCBI Transcriptome Shotgun Assembly (TSA) database accession numbers and miscellaneous sequence characteristics. 225 226 Danksagung 227
Danksagung
Prof. Dr. Ulrich Bathmann und Prof. Dr. Dieter Wolf-Gladrow danke ich ganz herzlich für die Begutachtung meiner Arbeit. Prof. Dr. Dietmar Blohm danke ich, dass er die
Rolle des dritten Prüfers übernimmt.
Danke an Dr. Uwe John und Prof. Dr. Allan Cembella für die Einwerbung von
Geldmitteln und die Bereitstellung meines Themas. Ein besonderes Danke an Uwe für die Betreuung und an Sára Beszteri für die gute, intensive Zusammenarbeit auch und gerade in den stressigsten Projektphasen.
Danke an Dr. Bánk Beszteri, Stefan Neuhaus, Stefan Pinkernell und Prof. Dr. Stephan
Frickenhaus von der AG Bioinformatik für die vielen kleinen und großen Hilfen bei der
Datenauswertung.
Danke an die aktuellen und ehemaligen Mitglieder der AG Cembella für die freundliche
Arbeitsatmosphäre und die vielen gemeinsamen Mittagspausen:
Dr. Tilman Alpermann, Chibo Chikwililwa, Wolgang Drebing, Dr. Anneli Ehlers,
Michael Freitag, Christel Hoffmann, Jennifer Hülskötter, Nina Jaeckisch, Eva Käppel,
Sascha Klöpper, Dr. Bernd Krock, Chishimba M.Kantu, Haiyan Ma, Ines Marschallek,
Annegret Müller, Yean-Sin Ong, Andrea Reents, Petra Steffens, Dr. Karina Stucken,
Dr. Urban Tillmann, Sylke Wohlrab und Birgit Wolters.
Danke an Steffi Gäbler, Maddalena Bayer, Linda Medlin und Jessica Kegel.
Special thanks to Dr. Thomas Richards.
Vielen Dank an meine Eltern, Stephi, und natürlich Chaoliang für Eure Unterstützung und Geduld.
This thesis was financed by EU-project ESTTAL (GOCE-CT2004-511154) and Marine
Genomics.